Synthesis, fungicidal activity, and sterol 14α-demethylase binding interaction of 2-azolyl-3,4-dihydroquinazolines on Penicillium digitatum

Article abstract of DOI:10.1021/jf305355u

A series of new 2-azolyl-3,4-dihydroquinazolines 6 was synthesized by direct cyclization of imidazole or 1,2,4-triazole with carbodiimides 4, which were obtained from aza-Wittig reaction of iminophosphorane 3 with isocyanate. The preliminary bioassay results demonstrated that most of the 2-imidazolyl-3,4-dihydroquinazolines 6a-6i exhibited good to significant fungicidal activity against Penicillium digitatum, whereas 2-triazolyl-3,4- dihydroquinazolines 6j-6t exhibited low fungicidal activity. Some of the 2-imidazolyl-3,4-dihydroquinazolines 6a-6i also exhibited strong binding interaction with the cytochrome P450-dependent sterol 14α-demethylase (CYP51). For example, compound 6e showed the best fungicidal activity against P. digitatum with IC₅₀ = 4.14 μg/mL and the best CYP51 binding activity with K_d = 0.34 μg/mL, both superior to those of the agricultural fungicide triadimefon.

Full text of DOI:10.1021/jf305355u

Article
pubs.acs.org/JAFC
Synthesis, Fungicidal Activity, and Sterol 14α-Demethylase Binding
Interaction of 2‑Azolyl-3,4-dihydroquinazolines on Penicillium
digitatum
Wen-Jin Li,†,∥ Qian Li,‡,∥ De-Li Liu,*,‡ and Ming-Wu Ding*,†
†Key Laboratory of Pesticide and Chemical Biology of the Ministry of Education and ‡Hubei Key Laboratory of Genetic Regulation
and Integrative Biology, College of Life Science, Central China Normal University, Wuhan 430079, China
S
* Supporting Information
ABSTRACT: A series of new 2-azolyl-3,4-dihydroquinazolines 6 was synthesized by direct cyclization of imidazole or 1,2,4-
triazole with carbodiimides 4, which were obtained from aza-Wittig reaction of iminophosphorane 3 with isocyanate. The
preliminary bioassay results demonstrated that most of the 2-imidazolyl-3,4-dihydroquinazolines 6a−6i exhibited good to
significant fungicidal activity against Penicillium digitatum, whereas 2-triazolyl-3,4-dihydroquinazolines 6j−6t exhibited low
fungicidal activity. Some of the 2-imidazolyl-3,4-dihydroquinazolines 6a−6i also exhibited strong binding interaction with the
cytochrome P450-dependent sterol 14α-demethylase (CYP51). For example, compound 6e showed the best fungicidal activity
against P. digitatum with IC50 = 4.14 μg/mL and the best CYP51 binding activity with Kd = 0.34 μg/mL, both superior to those
of the agricultural fungicide triadimefon.
KEYWORDS: imidazole, triazole, quinazoline, aza-Wittig reaction, fungicidal activity, 14α-demethylase, binding constant
eradicant power toward a wide spectrum of crop diseases.
Although various structure types of azoles have been
synthesized and evaluated as fungicides previously,11 the azole
compounds substituted by another heterocycle through N1 of
imidazole and the triazole ring were less investigated. Questions
concerning synthesis and fungicidal activities of new structures
of azole derivatives substituted by another heterocycle and the
binding ability of the cytochrome P450-dependent lanosterol
14α-demethylase (CYP51) for these azole fungicides are
therefore of particular interest in this study.
INTRODUCTION
■
Green mold of citrus, caused by Penicillium digitatum, is one of
the most serious diseases affecting postharvest citrus. Serious
postharvest losses are caused every year in most regions of the
world due to this pathogen infection.1,2 Synthetic fungicides are
widely utilized to control postharvest diseases in fruits and
vegetables. Among various kinds of fungicides, azoles are the
most widely used and studied class of antifungal agents in
agricultural usage because of their lower application doses, high
selectivity, and reduced undesired environmental impact.3,4 The
biochemical site of action of the fungicidal azoles is the
cytochrome P450-dependent lanosterol 14α-demethylase
(CYP51), encoded by the ERG11 gene in fungal cells.5 This
heme protein binds tetracyclic steroid molecules, inserting one
oxygen atom into a CH bond of the C-14 methyl group.6 Azole
antifungal agents inhibit CYP51 by a mechanism in which the
heterocyclic nitrogen atom (N3 of imidazole and N4 of
triazole) binds to the heme iron atom in the binding site of the
enzyme. Unfortunately, azoles are fungistatic against yeasts, and
the broad usage of these compounds led to the development of
resistance, showing the urgent need for new and effective
antifungal agents.
In previous studies,12−15 we have carried out cloning,
expression, and inhibition of CYP51 from P. digitatum. The
structural characteristics of the interaction between heterolo-
gous CYP51 and commercial azoles were also analyzed by
binding assay. Here we report an efficient synthesis of new
structures of 2-azolyl-3,4-dihydroquinazolines by direct cycliza-
tion of imidazole or 1,2,4-triazole with carbodiimides, which
were obtained from the aza-Wittig reaction of iminophosphor-
ane with isocyanate. Their fungicidal activity against P.
digitatum and binding interactions with the cytochrome P450-
dependent lanosterol 14α-demethylase (CYP51) are also
investigated.
Heterocyclic compounds play an increasingly important role
in pesticide discovery because many of them have shown good
insecticidal or fungicidal activities.7−10 Among them, 1,2,4-
triazole derivatives such as diniconazole, hexaconazole,
triadimefon, and triadimenol represent the most important
category of fungicides to date. Imidazole compounds exhibited
also siginificant fungicidal activities, and some of these
compounds have been utilized as agricultural fungicides. For
example, triadimefon, prochloraz, triflumizole, and pefurazoate
are widely used to control various fungal pathogens (Figure 1).
These compounds have excellent protective, curative, and
MATERIALS AND METHODS
■
Unless otherwise noted, all materials were commercially available and
were used directly without further purification. All solvents were
redistilled before use. P. digitatum was provided through the courtesy
of the Center for Bioassay, Central China Normal University. 1H
NMR spectra were recorded on a Mercury-Plus 400 or 600
Received: December 15, 2012
Revised: January 21, 2013
Accepted: January 25, 2013
© XXXX American Chemical Society
A
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Journal of Agricultural and Food Chemistry
Article
Figure 1. Some representative structures of agrocultural azole fungicides.
Scheme 1. General Synthetic Route for Compounds 6
spectrometer in CDCl3 or DMSO-d6 with TMS as the internal
reference. MS spectra were determined using a Trace MS 2000 organic
mass spectrometer. IR were recorded on a PE-983 infrared
spectrometer as KBr pellets with absorption in cm−1. Elemental
analyses were performed on a Vario EL III elemental analysis
instrument. Melting points were taken on an X-4 binocular microscope
melting point apparatus (Beijing Tech Instruments Co., Beijing,
China) and are uncorrected. The azides 2 were prepared according to
the reported method.16,17 The general synthesis of target compounds
is shown in Scheme 1, and the results are listed in Table 1.
reaction mixture was stirred for 4 h at ambient temperature, the
solvent was removed under reduced pressure, and the residual was
recrystallized from methylene dichloride/petroleum ether to give
iminophosphorane 3.
4-[2-(Triphenylphosphoranylideneamino)phenyl]but-3-en-2-one
1
(3a): yield, 94%; light yellow solid; mp, 119−121 °C; H NMR (400
MHz, CDCl3), δ 8.71 (d, J = 16.4 Hz, 1H, CH), 7.78−7.45 (m,
16H, ArH), 6.90 (t, J = 8.0 Hz, 1H, ArH), 6.72−6.63 (m, 2H,
ArH and CH), 6.47 (d, J = 8.4 Hz, 1H, ArH), 2.43 (s, 3H,
CH3); MS (70 eV), m/z (%) 421 (M+, 3), 378 (100), 277 (22), 143
(33), 108 (35). Anal. Calcd for C28H24NOP (421.5): C, 79.79; H,
5.74; N, 3.32. Found: C, 79.58; H, 5.83; N, 3.51.
General Procedure for Preparation of Iminophosphorane 3.
To a stirred solution of azide 2 (3 mmol) in anhydrous methylene
chloride (10 mL) was added dropwise triphenylphosphine (0.79 g, 3
mmol) in methylene chloride (10 mL) at room temperature. After the
4-[5-Chloro-2-(triphenylphosphoranylideneamino)phenyl]but-3-
1
en-2-one (3b): yield, 90%; light yellow solid; mp, 155−156 °C; H
NMR (600 MHz, CDCl3), δ 8.56 (d, J = 16.8 Hz, 1H, CH), 7.74−
7.45 (m, 16H, ArH), 6.83 (d, J = 6.0 Hz, 1H, ArH), 6.66 (d, J =
16.8 Hz, 1H, CH), 6.36 (d, J = 9.0 Hz, 1H, ArH), 2.41 (s, 3H,
CH3); MS (70 eV), m/z (%) 457 (M+, 8), 455 (23), 412 (94), 207
(97), 183 (100), 177 (70), 115 (30), 73 (37). Anal. Calcd for
C28H23ClNOP (455.9): C, 73.76; H, 5.08; N, 3.07. Found: C, 73.51;
H, 5.13; N, 3.21.
Table 1. Preparation of Compounds 6
a
compd
X
R1
R2
R3
yield (%)
6a
6b
6c
6d
6e
6f
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
Cl
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
H
H
Me
Me
Me
Me
Me
Me
OEt
OEt
Me
Me
OEt
OEt
Me
Me
Me
Me
Me
Me
t-Bu
t-Bu
i-Pr
i-Pr
85
86
80
72
77
73
77
83
74
68
62
75
87
86
91
69
62
84
84
81
1-[2-(Triphenylphosphoranylideneamino)phenyl]-4,4-dimethyl-
pent-1-en-3-one (3c): yield, 90%; light yellow solid; mp, 181−183 °C;
1H NMR (400 MHz, CDCl3), δ 8.69 (d, J = 16.0 Hz, 1H, CH),
n-Bu
4-ClC6H4
4-FC6H4
Ph
7.78−7.43 (m, 16H, ArH), 7.28 (d, J = 15.6 Hz, 1H, CH), 6.85
(t, J = 8.4 Hz, 1H, ArH), 6.63 (t, J = 7.6 Hz, 1H, ArH), 6.45 (d, J
= 8.4 Hz, 1H, ArH), 1.19 (s, 9H, 3CH3); MS (70 eV), m/z (%) 463
(M+, 4), 378 (80), 277 (55), 210 (49), 183 (95), 170 (100), 77 (49),
40 (54). Anal. Calcd for C31H30NOP (463.6): C, 80.32; H, 6.52; N,
3.02. Found: C, 80.58; H, 6.67; N, 3.08.
6g
6h
6i
Ph
4-FC6H4
4-FC6H4
Ph
6j
6k
6l
N
4-ClC6H4
4-FC6H4
i-Pr
Ethyl 3-[5-chloro-2-(triphenylphosphoranylideneamino)phenyl]-
1
acrylate (3d): yield, 85%; light yellow solid; mp, 171−172 °C; H
N
NMR (600 MHz, CDCl3), δ 8.64 (d, J = 16.2 Hz, 1H, CH), 7.75−
7.43 (m, 16H, ArH), 6.79 (d, J = 8.4 Hz, 1H, ArH), 6.44 (d, J =
16.8 Hz, 1H, CH), 6.35 (d, J = 8.4 Hz, 1H, ArH), 4.26 (q, J = 7.2
Hz, 2H, OCH2), 1.36 (t, J = 7.2 Hz, 3H, CH3); MS (70 eV), m/z (%)
485 (M+, 3), 412 (66), 262 (25), 183 (100), 108 (57), 44 (53). Anal.
Calcd for C29H25ClNO2P (485.9): C, 71.68; H, 5.19; N, 2.88. Found:
C, 71.94; H, 5.01; N, 2.89.
6m
6n
6o
6p
6q
6r
6s
6t
N
N
Ph
N
3-MeC6H4
4-ClC6H4
4-FC6H4
i-Pr
N
N
N
N
Ph
General Procedure for Preparation of 2-Azolyl-3,4-dihydro-
quinazolines 6. To a solution of iminophosphorane 3 (2 mmol) in
anhydrous methylene chloride (10 mL) was added isocyanate R3NCO
(2 mmol). After the reaction mixture was stirred at room temperature
N
3-MeC6H4
a
Isolated yields based on iminophosphorane 3.
B
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Journal of Agricultural and Food Chemistry
Article
for 2−4 h (R3 is an aryl group) or refluxed for 8−12 h (R3 is an alkyl
group), the solvent was removed under reduced pressure and ether/
petroleum ether (1:2, 20 mL) was added to precipitate triphenyl-
phosphine oxide. Filtered, the solvent was removed to give
carbodiimide 4, which was used directly without further purification.
To the solution of 4 prepared above in CH3CN (10 mL) was added
imidazole (0.14 g, 2 mmol) and catalytic solid K2CO3 (0.03 g, 0.2
mmol). The mixture was stirred for 1−2 h at room temperature and
filtered, the filtrate was condensed, and the residual was recrystallized
from methylene dichloride/petroleum ether to give 2-azolyl-3,4-
dihydroquinazolines 6.
1-(3-Phenyl-3,4-dihydro-2-(1H-imidazol-1-yl)quinazolin-4-yl)-
1
propan-2-one (6f): white solid; mp, 158−160 °C; H NMR (400
MHz, CDCl3), δ 7.95 (s, 1H, imidazolyl-2-H), 7.42−6.97 (m, 11H,
Ar−H), 5.41 (t, J = 7.2 Hz, 1H, CH), 3.10 (dd, J1 = 7.2 Hz, J2 = 16.8
Hz, 1H, CHCO), 2.84 (dd, J1 = 6.4 Hz, J2 = 16.8 Hz, 1H, CHCO),
2.15 (s, 3H, CH3); 13C NMR (150 MHz, CDCl3), δ 205.5, 143.4,
140.4, 136.9, 129.9, 129.6, 128.7, 126.5, 126.4, 125.7, 125.2, 124.4,
122.5, 117.7, 59.1, 48.6, 31.3; MS (70 eV), m/z (%) 330 (M+, 3), 273
(100), 210 (36), 143 (21), 91 (16), 77 (59), 68 (23). Anal. Calcd for
C20H18N4O (330.4): C, 72.71; H, 5.49; N, 16.96. Found: C, 72.79; H,
5.21; N, 16.83.
Ethyl 2-(3,4-dihydro-2-(1H-imidazol-1-yl)-3-phenylquinazolin-4-
1-(6-Chloro-3,4-dihydro-2-(1H-imidazol-1-yl)-3-isopropylquina-
1
1
yl)acetate (6g): white solid; mp, 44−46 °C; H NMR (600 MHz,
zolin-4-yl)propan-2-one (6a): white solid; mp, 97−99 °C; H NMR
CDCl3), δ 7.96 (s, 1H, imidazolyl-2-H), 7.43−6.97 (m, 11H, Ar−H),
5.32 (dd, J1 = 5.4 Hz, J2 = 9.0 Hz, 1H, CH), 4.23 (q, J = 7.2 Hz, 2H,
OCH2), 2.88 (dd, J1 = 9.6 Hz, J2 = 15.0 Hz, 1H, CHCO), 2.65 (dd, J1
= 5.4 Hz, J2 = 15.0 Hz, 1H, CHCO), 1.26 (t, J = 7.2 Hz, 3H, CH3).;
13C NMR (100 MHz, CDCl3), δ 170.3, 143.3, 143.0, 140.3, 136.9,
129.7, 129.6, 128.8, 126.4, 125.9, 125.6, 125.0, 124.5, 122.3, 117.7,
61.1, 60.6, 40.6, 14.0; MS (70 eV), m/z (%) 360 (M+, 35), 272 (100),
218 (25), 128 (14), 77 (66), 51 (14). Anal. Calcd for C21H20N4O2
(360.4): C, 69.98; H, 5.59; N, 15.55. Found: C, 69.81; H, 5.47; N,
15.72.
(600 MHz, CDCl3), δ 8.04 (s, 1H, imidazolyl-2-H), 7.37 (s, 1H, Ar−
H), 7.28−7.17 (m, 4H, Ar−H), 5.07 (dd, J1 = 5.4 Hz, J2 = 7.8 Hz, 1H,
CH), 3.78−3.74 (m, 1H, NCH), 2.82 (dd, J1 = 8.4 Hz, J2 = 17.4 Hz,
1H, CHCO), 2.64 (dd, J1 = 5.4 Hz, J2 = 16.8 Hz, 1H, CHCO), 2.08 (s,
3H, CH3), 1.34 (d, J = 6.0 Hz, 3H, CH3), 0.97 (d, J = 6.6 Hz, 3H,
CH3); 13C NMR (100 MHz, CDCl3), δ 204.9, 146.7, 139.9, 136.6,
131.0, 130.4, 128.9, 128.6, 125.3, 124.9, 117.7, 52.1, 49.5, 48.0, 31.2,
22.1, 20.9; MS (70 eV), m/z (%) 330 (M+, 6), 273 (46), 231 (100),
177 (23), 163 (16), 68 (34), 43 (50). Anal. Calcd for C17H19ClN4O
(330.8): C, 61.72; H, 5.79; N, 16.94. Found: C, 61.58; H, 5.68; N,
16.96.
1-(3,4-Dihydro-2-(1H-imidazol-1-yl)-3-isopropylquinazolin-4-yl)-
propan-2-one (6b): white solid; mp, 61−63 °C; 1H NMR (400 MHz,
CDCl3), δ 8.07 (s, 1H, imidazolyl-2-H), 7.40−7.16 (m, 6H, Ar−H),
5.11 (dd, J1 = 5.2 Hz, J2 = 7.6 Hz, 1H, CH), 3.78−3.72 (m, 1H,
NCH), 2.82 (dd, J1 = 8.0 Hz, J2 = 16.4 Hz, 1H, CHCO), 2.65 (dd, J1 =
5.6 Hz, J2 = 16.4 Hz, 1H, CHCO), 2.03 (s, 3H, CH3), 1.36 (d, J = 6.8
Hz, 3H, CH3), 0.97 (d, J = 6.8 Hz, 3H, CH3); 13C NMR (100 MHz,
CDCl3), δ 205.3, 146.3, 141.0, 136.5, 130.0, 128.4, 127.3, 126.0, 124.7,
123.9, 117.7, 51.7, 49.4, 48.4, 31.2, 21.9, 20.7; MS (70 eV), m/z (%)
296 (M+, 3), 239 (49), 197 (100), 143 (22), 102 (15), 68 (24), 40
(35). Anal. Calcd for C17H20N4O (296.4): C, 68.89; H, 6.80; N, 18.90.
Found: C, 68.54; H, 6.92; N, 18.78.
Ethyl 2-(3-(4-fluorophenyl)-3,4-dihydro-2-(1H-imidazol-1-yl)-
1
quinazolin-4-yl)acetate (6h): white solid; mp, 39−41 °C; H NMR
(600 MHz, CDCl3), δ 7.94 (s, 1H, imidazolyl-2-H), 7.43−6.90 (m,
10H, Ar−H), 5.24 (dd, J1 = 4.2 Hz, J2 = 9.6 Hz, 1H, CH), 4.24 (q, J =
7.2 Hz, 2H, OCH2), 2.86 (dd, J1 = 9.6 Hz, J2 = 15.0 Hz, 1H, CHCO),
2.62 (dd, J1 = 4.8 Hz, J2 = 15.6 Hz, 1H, CHCO), 1.28 (t, J = 7.2 Hz,
3H, CH3); 13C NMR (150 MHz, CDCl3), δ 170.5, 161.0, 159.3, 143.0,
140.1, 139.4, 136.9, 129.8, 128.9, 126.6, 125.6, 124.9, 124.5, 124.3,
117.7, 116.5, 116.4, 61.2, 60.9, 40.4, 14.0; MS (70 eV), m/z (%) 378
(M+, 5), 236 (13), 217 (12), 134 (26), 108 (21), 94 (100), 75 (80).
Anal. Calcd for C21H19FN4O2 (378.4): C, 66.66; H, 5.06; N, 14.81.
Found: C, 66.69; H, 5.21; N, 14.73.
1-(6-Chloro-3-(4-fluorophenyl)-3,4-dihydro-2-(1H-imidazol-1-yl)-
quinazolin-4-yl)propan-2-one (6i). white solid; mp, 146−147 °C; 1H
NMR (600 MHz, CDCl3), δ 7.93 (s, 1H, imidazolyl-2-H), 7.35−6.92
(m, 9H, Ar−H), 5.29−5.26 (m, 1H, CH), 3.12−3.08 (m, 1H,
CHCO), 2.78−2.74 (m, 1H, CHCO), 2.18 (s, 3H, CH3); 13C NMR
(150 MHz, CDCl3), δ 205.1, 161.1, 159.4, 143.5, 139.3, 138.9, 136.8,
131.5, 130.1, 128.8, 127.5, 125.6, 125.1, 124.5, 117.5, 116.6, 116.4,
58.8, 48.1, 31.1; MS (70 eV), m/z (%) 382 (M+, 5), 325 (100), 244
(33), 122 (13), 95 (27), 75 (9). Anal. Calcd for C20H16ClFN4O
(382.8): C, 62.75; H, 4.21; N, 14.64. Found: C, 62.74; H, 4.13; N,
14.83.
1-(6-Chloro-3,4-dihydro-3-phenyl-2-(1H-1,2,4-triazol-1-yl)-
quinazolin-4-yl)propan-2-one (6j): white solid; mp, 152−154 °C; 1H
NMR (600 MHz, CDCl3), δ 8.79 (s, 1H, triazolyl-5-H), 7.86 (s, 1H,
triazolyl-3-H), 7.38−7.13 (m, 6H, Ar−H), 6.94 (d, J = 7.2 Hz, 2H,
Ar−H), 5.45 (t, J = 6.6 Hz, 1H, CH), 3.17 (dd, J1 = 6.0 Hz, J2 = 17.4
Hz, 1H, CHCO), 2.95 (dd, J1 = 7.2 Hz, J2 = 17.4 Hz, 1H, CHCO),
2.19 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3), δ 205.2, 152.8,
144.6, 143.1, 142.8, 138.7, 132.0, 129.5, 128.9, 128.1, 126.0, 125.8,
125.6, 122.6, 58.6, 48.9, 31.4; MS (70 eV), m/z (%) 365 (M+, 6), 322
(45), 308 (100), 281 (23), 245 (21), 218 (18), 163 (21), 77 (70).
Anal. Calcd for C19H16ClN5O (365.8): C, 62.38; H, 4.41; N, 19.14.
Found: C, 62.40; H, 4.63; N, 19.02.
1-(3-Butyl-3,4-dihydro-2-(1H-imidazol-1-yl)quinazolin-4-yl)-
1
propan-2-one (6c): light yellow oil; H NMR (600 MHz, CDCl3), δ
7.98 (s, 1H, imidazolyl-2-H), 7.31−7.10 (m, 6H, Ar−H), 5.03−4.99
(m, 1H, CH), 3.41−3.37 (m, 1H, NCH), 2.96 (dd, J1 = 8.4 Hz, J2 =
17.4 Hz, 1H, CHCO), 2.65 (dd, J1 = 4.2 Hz, J2 = 12.6 Hz, 1H,
CHCO), 2.11 (s, 3H, CH3), 1.42−1.01 (m, 4H, CH2CH2), 0.69 (t, J =
7.2 Hz, 3H, CH3); 13C NMR (100 MHz, CDCl3), δ 205.7, 145.7,
141.3, 136.6, 129.7, 128.2, 125.9, 125.7, 124.1, 123.7, 117.8, 54.2, 51.5,
48.8, 31.4, 30.7, 19.1, 13.1; MS (70 eV), m/z (%) 310 (M+, 3), 253
(78), 197 (100), 170 (17), 143 (62), 129 (31), 68 (54). Anal. Calcd
for C18H22N4O (310.4): C, 69.65; H, 7.14; N, 18.05. Found: C, 69.37;
H, 7.38; N, 18.21.
1-(3-(4-Chlorophenyl)-3,4-dihydro-2-(1H-imidazol-1-yl)-
quinazolin-4-yl)propan-2-one (6d): white solid; mp, 111−113 °C; 1H
NMR (600 MHz, CDCl3), δ 7.94 (s, 1H, imidazolyl-2-H), 7.42−6.99
(m, 10H, Ar−H), 5.34 (t, J = 7.2 Hz, 1H, CH), 3.10 (dd, J1 = 7.8 Hz,
J2 = 17.4 Hz, 1H, CHCO), 2.76 (dd, J1 = 5.4 Hz, J2 = 17.4 Hz, 1H,
CHCO), 2.15 (s, 3H, CH3); 13C NMR (150 MHz, CDCl3), δ 205.6,
143.0, 142.0, 140.2, 136.8, 131.2, 130.2, 129.7, 128.9, 126.7, 126.3,
125.1, 124.5, 123.8, 117.6, 59.1, 48.3, 31.4; MS (70 eV), m/z (%) 364
(M+, 3), 307 (100), 210 (68), 153 (30), 143 (59), 128 (24), 111 (37),
68 (43). Anal. Calcd for C20H17ClN4O (364.8): C, 65.84; H, 4.70; N,
15.36. Found: C, 65.89; H, 4.92; N, 15.15.
1-(3-(4-Fluorophenyl)-3,4-dihydro-2-(1H-imidazol-1-yl)-
quinazolin-4-yl)propan-2-one (6e): white solid; mp, 139−141 °C; 1H
NMR (600 MHz, CDCl3), δ 7.94 (s, 1H, imidazolyl-2-H), 7.42−6.91
(m, 10H, Ar−H), 5.31 (t, J = 4.8 Hz, 1H, CH), 3.09 (dd, J1 = 9.0 Hz,
J2 = 17.4 Hz, 1H, CHCO), 2.76 (dd, J1 = 5.4 Hz, J2 = 17.4 Hz, 1H,
CHCO), 2.15 (s, 3H, CH3); 13C NMR (150 MHz, CDCl3), δ 205.6,
161.0, 159.4, 143.4, 140.3, 139.5, 136.7, 131.9, 130.0, 128.8, 128.3,
126.6, 126.1, 125.1, 124.5, 117.6, 116.5, 59.4, 48.4, 31.3; MS (70 eV),
m/z (%) 348 (M+, 5), 291 (100), 210 (41), 143 (31), 109 (16), 95
(29), 68 (27). Anal. Calcd for C20H17FN4O (348.4): C, 68.95; H, 4.92;
N, 16.08. Found: C, 68.71; H, 4.90; N, 16.31.
Ethyl 2-(6-chloro-3-(4-chlorophenyl)-3,4-dihydro-2-(1H-1,2,4-tri-
azol-1-yl)quinazolin-4-yl)acetate (6k): white solid; mp, 152−153
1
°C; H NMR (600 MHz, CDCl3), δ 8.76 (s, 1H, triazolyl-5-H), 7.89
(s, 1H, triazolyl-3-H), 7.41−7.14 (m, 5H, Ar−H), 6.95 (d, J = 8.4 Hz,
2H, Ar−H), 5.29 (t, J = 7.2 Hz, 1H, CH), 4.24−4.21 (m, 2H, OCH2),
2.93 (dd, J1 = 7.8 Hz, J2 = 15.6 Hz, 1H, CHCO), 2.74 (dd, J1 = 7.2 Hz,
J2 = 15.6 Hz, 1H, CHCO), 1.21 (t, J = 7.2 Hz, 3H, CH3); 13C NMR
(150 MHz, CDCl3), δ 169.8, 152.9, 144.5, 142.4, 141.3, 138.4, 132.1,
131.6, 129.5, 129.1, 127.2, 126.0, 125.2, 123.9, 61.3, 60.0, 40.4, 14.0;
MS (70 eV), m/z (%) 429 (M+, 9), 342 (100), 315 (17), 253 (20),
163 (20), 111 (37), 74 (34). Anal. Calcd for C20H17Cl2N5O2 (430.3):
C, 55.83; H, 3.98; N, 16.28. Found: C, 55.95; H, 3.87; N, 16.56.
C
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Journal of Agricultural and Food Chemistry
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Table 2. Fungicidal Activities and IC50 of Compounds 6 on Penicillium digitatum
inhibition under different concentrations
(%, μg/mL)
compd
6a
X
R1
R2
R3
50
30
25
10
5
1
toxicological eq
IC50 (μg/mL)
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
Cl
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
H
H
Me
Me
Me
Me
Me
Me
OEt
OEt
Me
Me
OEt
OEt
Me
Me
Me
Me
Me
Me
t-Bu
t-Bu
i-Pr
i-Pr
12
15
29
35
86
61
35
23
64
8
0
0
5
0
9
8
6
0
9
y = 0.0924x − 0.1066
y = 0.0954x − 0.1198
y = 0.2325x − 0.2881
y = 0.3211x − 0.4289
y = 0.3071x − 0.1498
y = 0.2664x − 0.1024
y = 0.2964x − 0.3376
y = 0.156x − 0.178
128
6b
28
100
100
100
100
100
61
4
14
14
52
29
20
14
21
126
6c
n-Bu
57
100
100
15.58
10.40
4.14
5.06
10.94
29.79
7.12
>50
6d
4-ClC6H4
4-FC6H4
Ph
6e
6f
6g
Ph
6h
4-FC6H4
4-FC6H4
Ph
48
6i
100
8
100
y = 0.3155x − 0.305
6j
6k
N
4-ClC6H4
4-FC6H4
i-Pr
6
>50
6l
N
4
>50
6m
N
17
>50
6n
N
Ph
24
11
19
0
0
0
y = 0.0844x − 0.1219
y = 0.137x − 0.1562
129
6o
N
3-MeC6H4
4-ClC6H4
4-FC6H4
i-Pr
44
12
60.06
>50
6p
N
10
6q
N
14
>50
6r
N
10
>50
6s
N
Ph
35
15
22
60
8
8
0
0
0
y = 0.1124x − 0.1351
y = 0.1337x − 0.1597
y = 0.3328x − 0.3642
89.95
67.08
7.72
6t
N
3-MeC6H4
40
triadimefon
100
100
27
Ethyl 2-(6-chloro-3-(4-fluorophenyl)-3,4-dihydro-2-(1H-1,2,4-tria-
zol-1-yl)quinazolin-4-yl)acetate (6l): white solid; mp, 128−130 °C;
1H NMR (600 MHz, CDCl3), δ 8.74 (s, 1H, triazolyl-5-H), 7.88 (s,
H), 7.46−7.06 (m, 5H, Ar−H), 6.91 (d, J = 8.0 Hz, 1H, Ar−H), 6.78
(s, 1H, Ar−H), 6.73 (d, J = 8.0 Hz, 1H, Ar−H), 5.46 (t, J = 7.2 Hz,
1H, CH), 3.15 (dd, J1 = 6.0 Hz, J2 = 15.2 Hz, 1H, CHCO), 2.96 (dd, J1
= 7.6 Hz, J2 = 15.6 Hz, 1H, CHCO), 2.24 (s, 3H, CH3), 2.15 (s, 3H,
CH3); 13C NMR (100 MHz, CDCl3), δ 205.7, 152.7, 144.6, 143.0,
142.9, 140.0, 139.5, 129.2, 128.8, 126.8, 126.7, 125.6, 124.6, 123.0,
119.7, 59.1, 49.0, 31.6, 21.3; MS (70 eV), m/z (%) 345 (M+, 6), 302
(28), 288 (100), 261 (18), 233 (13), 211 (27), 129 (18), 91 (31).
Anal. Calcd for C20H19N5O (345.4): C, 69.55; H, 5.54; N, 20.28.
Found: C, 69.78; H, 5.37; N, 20.26.
1-(3,4-Dihydro-3-(4-chlorophenyl)-2-(1H-1,2,4-triazol-1-yl)-
quinazolin-4-yl)propan-2-one (6p): white solid; mp, 134−135 °C; 1H
NMR (400 MHz, CDCl3), δ 8.84 (s, 1H, triazolyl-5-H), 7.88 (s, 1H,
triazolyl-3-H), 7.46−7.13 (m, 6H, Ar−H), 6.91 (d, J = 9.2 Hz, 2H,
Ar−H), 5.42 (t, J = 6.8 Hz, 1H, CH), 3.16 (dd, J1 = 6.4 Hz, J2 = 17.2
Hz, 1H, CHCO), 2.89 (dd, J1 = 7.2 Hz, J2 = 17.2 Hz, 1H, CHCO),
2.17 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3), δ 205.6, 152.8,
144.4, 142.5, 141.5, 139.7, 131.2, 129.4, 128.8, 127.0, 126.6, 125.3,
124.6, 123.8, 59.1, 48.6, 31.5; MS (70 eV), m/z (%) 365 (M+, 5), 322
(28), 308 (100), 281 (33), 253 (51), 197 (62), 143 (98), 129 (59), 90
(48), 68 (52). Anal. Calcd for C19H16ClN5O (365.8): C, 62.38; H,
4.41; N, 19.14. Found: C, 62.21; H, 4.20; N, 19.37.
1-(3,4-Dihydro-3-(4-fluorophenyl)-2-(1H-1,2,4-triazol-1-yl)-
quinazolin-4-yl)propan-2-one (6q): white solid; mp, 127−129 °C; 1H
NMR (400 MHz, CDCl3), δ 8.81 (s, 1H, triazolyl-5-H), 7.87 (s, 1H,
triazolyl-3-H), 7.46−6.89 (m, 6H, Ar−H), 6.91 (d, J = 9.2 Hz, 2H,
Ar−H), 5.42 (t, J = 6.8 Hz, 1H, CH), 3.16 (dd, J1 = 6.4 Hz, J2 = 17.2
Hz, 1H, CHCO), 2.89 (dd, J1 = 7.2 Hz, J2 = 17.2 Hz, 1H, CHCO),
2.17 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3), δ 205.8, 161.3,
159.6, 152.9, 144.5, 143.0, 140.0, 139.2, 128.9, 127.0, 126.6, 125.4,
124.9, 124.7, 116.3, 116.2, 59.5, 49.0, 31.6; MS (70 eV), m/z (%) 349
(M+, 7), 306 (36), 293 (22), 292 (100), 265 (33), 211 (28), 129 (22),
95 (37). Anal. Calcd for C19H16FN5O (349.4): C, 65.32; H, 4.62; N,
20.05. Found: C, 65.48; H, 4.84; N, 19.81.
1H, triazolyl-3-H), 7.41−6.91 (m, 7H, Ar−H), 5.26 (t, J = 7.2 Hz, 1H,
CH), 4.24−4.21 (m, 2H, OCH2), 2.93 (dd, J1 = 7.8 Hz, J2 = 15.0 Hz,
1H, CHCO), 2.74 (dd, J1 = 6.6 Hz, J2 = 15.6 Hz, 1H, CHCO), 1.26 (t,
J = 7.2 Hz, 3H, CH3); 13C NMR (150 MHz, CDCl3), δ 169.8, 161.2,
159.6, 152.8, 144.5, 142.8, 138.9, 138.5, 132.0, 129.1, 127.0, 126.0,
125.2, 124.9, 116.4, 116.2, 61.3, 60.2, 40.5, 14.0; MS (70 eV), m/z (%)
413 (M+, 11), 326 (100), 299 (18), 163 (15), 95 (43), 75 (17). Anal.
Calcd for C20H17ClFN5O2 (413.8): C, 58.05; H, 4.14; N, 16.92.
Found: C, 58.04; H, 4.33; N, 16.70.
1-(6-Chloro-3,4-dihydro-3-isopropyl-2-(1H-1,2,4-triazol-1-yl)-
quinazolin-4-yl)propan-2-one (6m): white solid; mp, 128−130 °C;
1H NMR (600 MHz, CDCl3), δ 8.87 (s, 1H, triazolyl-5-H), 8.10 (s,
1H, triazolyl-3-H), 7.28−7.18 (m, 3H, Ar−H), 5.17 (dd, J1 = 4.2 Hz, J2
= 9.0 Hz, 1H, CH), 3.95−3.90 (m, 1H, NCH), 2.93 (dd, J1 = 9.6 Hz,
J2 = 16.8 Hz, 1H, CHCO), 2.69 (dd, J1 = 4.2 Hz, J2 = 17.4 Hz, 1H,
CHCO), 2.07 (s, 3H, CH3), 1.39 (d, J = 6.6 Hz, 3H, CH3), 1.08 (d, J =
7.2 Hz, 3H, CH3); 13C NMR (100 MHz, CDCl3), δ 205.3, 153.0,
145.8, 145.0, 140.0, 130.8, 128.8, 128.6, 125.1, 125.0, 51.6, 49.5, 48.0,
31.2, 21.9, 20.9; MS (70 eV), m/z (%) 331 (M+, 4), 274 (28), 232
(100), 205 (23), 163 (16), 43 (25). Anal. Calcd for C16H18ClN5O
(331.8): C, 57.92; H, 5.47; N, 21.11. Found: C, 57.93; H, 5.25; N,
21.38.
1-(3,4-Dihydro-3-phenyl-2-(1H-1,2,4-triazol-1-yl)quinazolin-4-yl)-
1
propan-2-one (6n): white solid; mp, 165−166 °C; H NMR (400
MHz, CDCl3), δ 8.81 (s, 1H, triazolyl-5-H), 7.87 (s, 1H, triazolyl-3-
H), 7.47−7.09 (m, 7H, Ar−H), 6.96 (d, J = 8.0 Hz, 2H, Ar−H), 5.49
(t, J = 7.2 Hz, 1H, CH), 3.17 (dd, J1 = 6.0 Hz, J2 = 16.8 Hz, 1H,
CHCO), 2.95 (dd, J1 = 7.6 Hz, J2 = 16.8 Hz, 1H, CHCO), 2.16 (s, 3H,
CH3); 13C NMR (100 MHz, CDCl3), δ 205.8, 152.7, 144.6 143.0,
142.9, 140.0, 129.4, 128.8, 126.9, 126.8, 125.8, 125.5, 124.6, 122.5,
59.1, 49.0, 31.6; MS (70 eV), m/z (%) 331 (M+, 6), 288 (26), 274
(100), 253 (31), 247 (25), 219 (25), 197 (36), 143 (43), 129 (38), 77
(67). Anal. Calcd for C19H17N5O (331.4): C, 68.87; H, 5.17; N, 21.13.
Found: C, 68.96; H, 5.02; N, 21.15.
1-(3,4-Dihydro-3-isopropyl-2-(1H-1,2,4-triazol-1-yl)quinazolin-4-
1
yl)propan-2-one (6r). white solid; mp, 87−89 °C; H NMR (600
MHz, CDCl3), δ 8.89 (s, 1H, triazolyl-5-H), 8.10 (s, 1H, triazolyl-3-
H), 7.31−7.15 (m, 4H, Ar−H), 5.20 (t, J = 4.8 Hz, 1H, CH), 3.91−
3.88 (m, 1H, NCH), 2.94 (dd, J1 = 9.0 Hz, J2 = 16.2 Hz, 1H, CHCO),
2.68 (dd, J1 = 3.6 Hz, J2 = 16.8 Hz, 1H, CHCO), 2.03 (s, 3H, CH3),
1.41 (d, J = 6.6 Hz, 3H, CH3), 1.07 (d, J = 6.6 Hz, 3H, CH3); 13C
1-(3,4-Dihydro-3-m-tolyl-2-(1H-1,2,4-triazol-1-yl)quinazolin-4-
1
yl)propan-2-one (6o): white solid; mp, 163−164 °C; H NMR (400
MHz, CDCl3), δ 8.79 (s, 1H, triazolyl-5-H), 7.88 (s, 1H, triazolyl-3-
D
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NMR (150 MHz, CDCl3), δ 205.7, 152.8, 145.4, 144.8, 140.1, 128.3,
127.2, 125.8, 125.0, 123.6, 51.2, 49.3, 48.3, 31.4, 21.7, 20.8; MS (70
eV), m/z (%) 297 (M+, 3), 240 (35), 198 (100), 171 (26), 129 (18).
Anal. Calcd for C16H19N5O (297.4): C, 64.63; H, 6.44; N, 23.55.
Found: C, 64.37; H, 6.68; N, 23.45.
1-(3,4-Dihydro-3-phenyl-2-(1H-1,2,4-triazol-1-yl)quinazolin-4-yl)-
3,3-dimethylbutan-2-one (6s). white solid; mp, 146−147 °C; 1H
NMR (400 MHz, CDCl3), δ 8.79 (s, 1H, triazolyl-5-H), 7.86 (s, 1H,
triazolyl-3-H), 7.46−7.01 (m, 7H, Ar−H), 6.99 (d, J = 7.6 Hz, 1H,
Ar−H), 5.56 (t, J = 7.6 Hz, 1H, Ar−H), 3.08 (d, J = 7.6 Hz, 2H,
CH2CO), 0.99 (s, 9H, 3CH3); 13C NMR (150 MHz, CDCl3), δ 212.1,
152.6, 144.4, 143.0, 142.8, 139.8, 129.3, 128.6, 126.7, 126.6, 125.6,
125.4, 124.4, 122.4, 59.3, 44.4, 42.2, 25.3.; MS (70 eV), m/z (%) 373
(M+, 3), 288 (70), 274 (100), 247 (19), 219 (15), 129 (14), 77 (26).
Anal. Calcd for C22H23N5O (373.5): C, 70.76; H, 6.21; N, 18.75.
Found: C, 70.94; H, 6.14; N, 18.84.
Determination of Protein Content. The protein concentration
was determined according to the Bradford method using bovine serum
albumin (BSA) as the protein standard.22
Determination of CYP51 Activity and Binding Spectrum
Analysis. According to the methods described in the literature,23 the
CYP51 content and the activity were determined. The solutions of
CYP51 were prepared with 100 mM PBS (pH 7.2), and UV−visible
absorption spectra were recorded for the oxidized species on a UV-240
IPC spectrophotometer at 25 °C. CYP51 solutions were reduced using
a small amount of sodium dithionite and bubbled briefly with carbon
monoxide to form the P450−CO complex and measure the precise
P450 concentration at 25 °C.24 The purified soluble PdCYP51 was
diluted to appropriate concentration and filled into a cuvette. After its
baseline was scanned at 350−500 nm on an S-3100 spectropho-
tometer, the drug was added to the cuvette. After staying for 1 min
under room temperature, the absorption spectrum of the mixture was
recorded (concentration of DMSO in the mixture was maintained at
<0.5%). The binding constant Kd was calculated by using the following
equation and Hanes−Woolf25 chart [I] versus [I]/ΔA:
1-(3,4-Dihydro-3-m-tolyl-2-(1H-1,2,4-triazol-1-yl)quinazolin-4-
1
yl)-3,3-dimethylbutan-2-one (6t): white solid; mp, 185−187 °C; H
NMR (400 MHz, CDCl3), δ 8.77 (s, 1H, triazolyl-5-H), 7.88 (s, 1H,
triazolyl-3-H), 7.46−6.76 (m, 8H, Ar−H), 5.54 (t, J = 7.0 Hz, 1H,
CH), 3.08 (dd, J1 = 8.4 Hz, J2 = 13.6 Hz, 1H, CHCO), 2.24 (s, 3H,
CH3), 0.96 (s, 9H, 3CH3); 13C NMR (100 MHz, CDCl3), δ 212.2,
152.6, 144.5, 143.1, 140.0, 139.5, 129.1, 128.7, 126.8, 126.6, 125.6,
124.5, 123.0, 119.7, 59.5, 44.5, 42.3, 25.4, 21.3; MS (70 eV), m/z (%)
387 (M+, 3), 302 (63), 288 (100), 261 (17), 171 (13), 129 (14), 91
(19). Anal. Calcd for C23H25N5O (387.5): C, 71.29; H, 6.50; N, 18.07.
Found: C, 71.17; H, 6.51; N, 18.31.
A = Amax[I]/(Kd + [I])
In the above equation, A is the value of the maximum, subtracting the
minimum observed shift in absorption at different drug concen-
trations; Amax is the value of the maximum, subtracting the minimum
observed shift in absorption at saturation; [I] is the drug
concentration; and Kd is the binding constant.
Fungicidal Activity Determination (Growth Inhibition Test).
The in vitro fungicidal activities against P. digitatum were tested
according to the reported method.18,19 The medium was amended
with aliquots of each tested compound solution to provide a
concentration of 50 mg/L. The tested compounds were dissolved in
0.3 mL of dimethyl sulfoxide (DMSO) and added aseptically to
molten agar after autoclaving, when the agar had cooled to
approximately 45−50 °C. The concentration of solvent never
exceeded 0.1 mg/L. The mixed medium without sample was used as
the blank control. The inocula, 5 mm in diameter, were removed from
the margins of actively growing colonies of mycelium, placed in the
centers of the above plates. Three replicates were done for each
concentration, and the control plates were sealed with parafilm and
incubated at 26 °C in darkness. The diameter of the mycelium was
measured after 48 h. The inhibition percent was used to describe the
control efficiency of the compounds. Inhibition percent (%) = (hyphal
diameter in the control − hyphal diameter in the treatment)/hyphal
diameter in the control. The results are summarized in Table 2.
Preparation of the Heterogeneous Expressed CYP51
Protein of P. digitatum. Cloning and expression of PdCYP51
were carried out by using reverse transcription PCR (RT-PCR) with
the purified mRNA as a template isolated from P. digitatum.12 The
amplified PdCYP51 cDNA fragment was cloned into pMD-T vector
for sequencing and then subcloned into pET-28 for expression. The
recombinant plasmid pET-PdCYP51 was transformed into Escherichia
coli BL21 (DE3), and the positive colonies were selected. The
expression of recombinant protein in E. coli BL21 (DE3) was induced
by 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 6 h at 25
°C before harvesting at 5000g for 10 min. After harvesting, cells were
washed twice with 100 mM phosphate-buffered saline (PBS) and
suspended in buffer A (50 mM KH2PO4, 50 mM K2HPO4, 20%
glycerol, 1 mM EDTA, 0.5% Triton X-100) with 1 mM dithiothreitol
(DTT) and 1 mM PMSF. The cells were lysed by sonication on ice, in
a UP200S cell disruptor at an intensity of 60 W for 5 min, with a 30 s
rest period between each burst. The lysate was centrifuged at 15000g
(30 min, 4 °C), and the supernatant containing soluble cellular
material was purified using the Ni-NTA affinity resin according to the
manufacturer’s protocol with certain modifications.20 All of the buffers
were adjusted to pH 7.2. The purified protein was dialyzed into 10
mM PBS (pH 7.2) to desalt, concentrated, and stored at −80 °C until
use.21
RESULTS AND DISCUSSION
■
Synthesis of Compounds 6. Many 1-substituted
imidazole and 1,2,4-triazole compounds are generally prepared
by N-alkylations of imidazole and 1,2,4-triazole with alkylating
reagents; however, many of these reactions are limited by the
simultaneous formation of the unwanted and biologically
inactive 2-isomer of imidazoles or 4-isomer of 1,2,4-triazoles.26
It was reported that 1,2,4-triazole derivatives can be
regiospecifically synthesized by addition reactions of 1,2,4-
triazole with aldehydes or carbodiimide, and this method was
successfully utilized to prepare many novel triazole deriva-
tives.27−29 The aza-Wittig reaction of iminophosphorane
provides an efficient way to synthesize various hetero-
cycles.30−33 It has been reported that quinazolines may be
prepared efficiently by tandem aza-Wittig reaction of a suitable
iminophosphorane, isocyanate, and various nucleophiles.17
Cyclization was achieved by initial nucleophilic addition of
various nucleophiles to the carbodiimide intermediate and
further intramolecular Michael addition. We envisioned that 2-
azolyl-3,4-dihydroquinazolines would be prepared directly if
imidazole and 1,2,4-triazole were used as the nucleophile.
As shown in Scheme 1, iminophosphoranes 3, obtained from
Staudinger reaction of azides 2 with triphenylphosphine,
reacted with isocyanates to give the carbodiimides 4 in
anhydrous methylene chloride at room temperature or under
reflux. Then carbodiimides 4 were allowed to react with
imidazole or 1H-1,2,4-triazole to provide 2-azolyl-3,4-dihydro-
quinazolines 6 in the presence of a catalytic amount of solid
potassium carbonate in 62−91% yields (Table 1). It is
noteworthy that the reaction proceeds under mild conditions
to give 2-azolyl-3,4-dihydroquinazolines 6, and the overall
transformation is realized in a simple one-pot procedure from
iminophosphoranes 3 in good overall yields. The formation of
6 can be viewed as an initial nucleophilic addition of imidazole
or 1H-1,2,4-triazole under potassium carbonate to give the
intermediate 5, which directly cyclized to give 6 through
intramolecular Michael addition.
E
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Journal of Agricultural and Food Chemistry
Article
Bioassay Activity of Compounds 6. The fungicidal
activities of compounds 6 were evaluated in vitro using agar
diffusion and broth dilution assay, the results of which are
presented in Table 2. Some of the compounds showed
significant activity in the initial screening against P. digitatum
cultures when tested at 50 μg/mL concentration using agar
diffusion assay. For example, compounds 6c, 6d, 6e, 6f, 6g, and
6i all exhibited 100% inhibitive activity. At lower (25 μg/mL)
concentration, compounds 6d, 6e, and 6i still showed 100%
inhibitive activity against P. digitatum. The fungicidal activities
of some compounds 6 were further determined at lower
concentrations (10, 5, and 1 μg/mL). The toxicological
equation and IC50 of the compounds were then obtained,
and the results are listed in Table 2.
As indicated in Table 2, the structure−activity relationships
clearly suggest that 2-triazolyl-substituted 3,4-dihydroquinazo-
lines 6 (compounds 6j−6t) show low fungicidal activity against
P. digitatum with 4−44% inhibition at 50 μg/mL and all with
IC50 > 50 μg/mL, whereas most of the 2-imidazolyl-substituted
3,4-dihydroquinazolines 6 (compounds 6c−6i) exhibited good
fungicidal activity except of compounds 6a and 6b.
Figure 2. CYP51 type II binding spectrum in the presence of
compound 6e. Type II spectral response to 6e and the concentrations
of the 6e added to the reaction mixture were 0.50, 0.75, 1.00, 1.25,
1.50, 2.00, and 2.50 μg/mL.
The fungicidal activity of imidazoles 6a−6i is obviously
related to R3 substituents. When R3 is an aromatic substituent,
good to excellent activity was reached, and the best three
compounds (6e, 6f, and 6i) have the smallest IC50 values (4.14,
5.06, and 7.12 μg/mL). The presence of a group such as 4-F
(6e and 6i) substitution on the phenyl ring also plays a
significant role in imparting antifungal activity to the
compounds. As R3 is an alkyl substituent, good activity was
obtained when R3 is a butyl group (6c), whereas low activities
resulted in the case that R3 is an isopropyl group (6a and 6b).
When R2 is a methyl group, better activity is generally obtained
than when R is a methoxy group. For example, compounds 6e
and 6f (R2 = Me) displayed better fungicidal activities than
compounds 6g and 6h (R2 = OEt) did.
Triadimefon, a broad-spectrum fungicide, was used as a
reference to study the activity of the potential compounds 6. As
indicated in Table 2, compound 6i showed in vitro activity
(IC50 = 7.12 μg/mL) as compared to triadimefon (IC50 = 7.72
μg/mL). Two compoundst 6e and 6f had comparably high
inhibitory activity (6e, IC50 = 4.14 μg/mL; 6f, IC50 = 5.06 μg/
mL) as compared to triadimefon against P. digitatum.
Figure 3. CYP51 type II binding spectrum in the presence of
compound 6i. Type II spectral response to 6i and the concentrations
of the 6i added to the reaction mixture were 0.50, 0.75, 1.00, 1.25,
1.50, 2.00, and 2.50 μg/mL.
Binding Spectrum Analysis of Compounds 6 with
PdCYP51. The binding interactions of some 2-imidazolyl-3,4-
dihydroquinazolines 6c, 6d, 6e, 6h and 6i with PdCYP51 were
investigated with the commercial fungicide triadimefon as
standard. UV−visible absorption spectroscopy provided a
simple and accurate method for the determination of the
level of binding of substrates and inhibitors to P450s. Similar to
our previous results, compounds 6c, 6d, 6e and 6i induced type
II binding spectra with a maximum absorbance at 415−420 nm
and a minimum at 390−400 nm (Figures 2 and 3), indicating a
shift in the heme iron of the cytochrome P450 to a low-spin
state on inhibitor binding. However, compound 6h induced
type I binding spectra with a maximum absorbance at 400 nm
and a minimum at 420 nm (Figure 4), which might be due to
the special influence of the ester group of compound 6h. The
magnitude of the spectral response increased steadily with
increasing fungicide concentration and attained saturation
when an equilibrium concentration of drug was added to the
PdCYP51 fractions. These spectra most likely resulted from an
interaction of the imidazole N-3 of 6 with the heme of the P450
causing a shift toward the high-spin state. This exhibited the
Figure 4. CYP51 type I binding spectrum in the presence of
compound 6h. Type I spectral response to 6h and the concentrations
of 6h added to the reaction mixture were 0.50, 0.75, 1.00, 1.25, 1.50,
2.00, and 2.50 μg/mL.
F
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Journal of Agricultural and Food Chemistry
Article
displacement of the native sixth ligand of the heme iron by the
nitrogen atom in the imidazole ring of 6.
The spectral results showed that the affinity of most of the
compounds 6 with the purified PdCYP51 was tight. The Kd
values of compounds 6c, 6d, 6e, 6h, and 6i were calculated to
be 4.13, 1.06, 0.34, 1.79, and 0.92 μg/mL, respectively (Table
3). The five imidazoles 6 exhibited a high and gradually
lanosterol 14α-demethylase. We are currently looking further
into the biological activities of this type of compound on other
plant pathogenic fungi.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H NMR for intermediates 3a−3d and 1H NMR and 13C NMR
spectra for compounds 6a−6t. This material is available free of
charge via the Internet at http://pubs.acs.org.
Table 3. In Vitro Binding Constants (Kd) and IC50 of Some
Compounds 6
AUTHOR INFORMATION
Corresponding Author
compd
■
6c
6d
6e
6h
6i
triadimefon
*Phone: 86-27-67867958. E-mail: ding5229@yahoo.com.cn or
deliliu2002@yahoo.com.cn.
Kd (μM)
4.13
1.06
0.34
4.14
1.79
0.92
7.12
0.42
7.72
IC50 (μg/mL)
15.58
10.40
29.79
Author Contributions
∥W.-J.L. and Q.L. are first coauthors and contributed equally to
this work.
weakening affinity for PdCYP51 in the order 6e > 6i > 6d > 6h
> 6c. Although compounds 6c, 6d, 6h, and 6i displayed lower
binding activities than triadimefon, compound 6e (Kd = 0.34
μg/mL) did exhibit stronger binding activities than triadimefon
(Kd = 0.42 μg/mL). This is consistent with the fact that
compound 6e displays a better fungicidal activity (IC50 = 4.14
μg/mL) against P. digitatum than triadimefon does (IC50 = 7.72
μg/mL).
Docking Model of Compound 6e with PdCYP51. On
the basis of our previous homology modeling of PdCYP51,12
compound 6e was docked in the active site with the N3 of the
imidazole ring forming a coordinate bond with the Fe of heme
(Figure 5). In the binding model, the distance between the N
Funding
We gratefully acknowledge financial support of this work by the
National Natural Science Foundation of China (No. 31071653,
21032001, 21172085).
Notes
The authors declare no competing financial interest.
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