Design, Synthesis, Crystal Structure, and Antimicrobial Evaluation of 6-Fluoroquinazolinylpiperidinyl-Containing 1,2,4-Triazole Mannich Base Derivatives against Phytopathogenic Bacteria and Fungi

Article abstract of DOI:10.1021/acs.jafc.0c01365

A total of 20 1,2,4-triazole Mannich base derivatives bearing the 6-fluoroquinazolinylpiperidinyl moiety were designed, synthesized, and evaluated as antimicrobial agents against phytopathogenic bacteria and fungi according to the molecular hybridization strategy. Of note, the structure of target compound 4h was clearly confirmed through single-crystal X-ray diffraction analysis. The turbidimetric assays indicated that some compounds exhibited excellent antibacterial efficacies in vitro against Xanthomonas oryzae pv. oryzae (Xoo). For example, compounds 4c, 4f, 4j, and 7j had EC50 values of 23.6, 18.8, 23.4, and 24.3 μg/mL, respectively, which were far superior to that of agrobactericide bismerthiazol (EC50 = 92.4 μg/mL). In particular, compound 4f demonstrated a potent anti-Xoo activity approximately five times more active than that of bismerthiazol. Moreover, in vivo assays showed the excellent protective and curative activities of compound 4f against rice bacterial blight, having the potential as an alternative bactericide for controlling Xoo. The structure-activity relationship analysis showed a good pesticide-likeness concerning compound 4f, following Tice's criteria. The anti-Xoo mechanism of compound 4f was preliminarily explored by scanning electron microscopy measurements in living bacteria. Finally, several compounds also exhibited good antifungal activities in vitro against Gibberella zeae at 50 μg/mL. In short, the presented work showed the potential of 6-fluoroquinazolinylpiperidinyl-containing 1,2,4-triazole Mannich base derivatives as effective bactericides for controlling Xoo.

Full text of DOI:10.1021/acs.jafc.0c01365

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Agricultural and Environmental Chemistry
Design, Synthesis, Crystal Structure, and Antimicrobial Evaluation
of 6-Fluoroquinazolinylpiperidinyl-Containing 1,2,4-Triazole Mannich
Base Derivatives against Phytopathogenic Bacteria and Fungi
Jun Shi, Muhan Ding, Na Luo, Suran Wan, Peijia Li, Junhong Li, and Xiaoping Bao
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.0c01365 • Publication Date (Web): 11 Aug 2020
Downloaded from pubs.acs.org on August 12, 2020
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Design, Synthesis, Crystal Structure, and Antimicrobial
Evaluation
of
6-Fluoroquinazolinylpiperidinyl-Containing
Mannich Base Derivatives against
1,2,4-Triazole
Phytopathogenic Bacteria and Fungi
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6
Jun Shi, Muhan Ding, Na Luo, Suran Wan, Peijia Li, Junhong Li, and Xiaoping Bao*
State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key
Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Centre for
Research and Development of Fine Chemicals, Guizhou University, Guiyang 550025, P. R. China
*Corresponding author: Tel.: +86(0851)-88292090; Fax: +86(0851)-83622211; E-mail:
baoxp_1980@aliyun.com
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Abstract: A total of twenty 1,2,4-triazole Mannich base derivatives bearing the
6-fluoroquinazolinylpiperidinyl moiety were designed, synthesized, and evaluated as
antimicrobial agents against phytopathogenic bacteria and fungi, according to
molecular hybridization strategy. Of note, the structure of target compound 4h was
clearly confirmed through single crystal X-ray diffraction analysis. The turbidimetric
assays indicated that some compounds exhibited excellent antibacterial efficacies in
vitro against Xanthomonas oryzae pv. oryzae (Xoo). For example, compounds 4c, 4f,
4j, and 7j had EC50 values of 23.6, 18.8, 23.4, and 24.3 μg/mL, respectively, far
superior to that of agrobactericide bismerthiazol (EC50 = 92.4 μg/mL). Particularly,
compound 4f demonstrated a potent anti-Xoo activity, approximately 5-fold more
active than bismerthiazol. Moreover, in vivo assays showed excellent protective and
curative activities of compound 4f against rice bacterial blight, having the potential as
an alternative bactericide for controlling Xoo. The structure-activity relationship
analysis showed a good pesticide-likeness concerning compound 4f, following Tice’s
criteria. The anti-Xoo mechanism of compound 4f was preliminarily explored by SEM
measurements in living bacteria. Finally, several compounds also exhibited good
antifungal activities in vitro against Gibberella zeae at 50 μg/mL. In short, the
presented work showed the potential of 6-fluoroquinazolinylpiperidinyl-containing
1,2,4-triazole Mannich base derivatives as effective bactericides for controlling Xoo.
Keywords: 6-fluoroquinazoline, 1,2,4-triazole Mannich base, antimicrobial
evaluation, structure-activity relationship (SAR)
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1. Introduction
Plant bacterial diseases lead to enormous yield and economic losses in agricultural
production every year.13 For example, rice bacterial leaf blight caused by
Xanthomonas oryzae pv. oryzae (Xoo) is considered as one of the most destructive
bacterial diseases that can adversely affect rice plants during the whole growth stage.
The pathogen of Xoo infects rice leaves mainly through the hydathodes at the leaf tip,
leaf margins as well as the wounded leaves or roots, colonizes the intercellular spaces
and enters the xylem vessels.1 It had been reported that rice bacterial blight reduced
the rice yield by ~50% in some Asian countries.4 Of note, the pathogen of Xoo ranked
4th on the list of top 10 plant pathogenic bacteria because of its particular significance
in agriculture.1 Moreover, citrus canker caused by Xanthomonas axonopodis pv. citri
(Xac) is a devastating bacterial disease, which leads to lesions on the leaves, fruits &
stems and thus gives rise to serious damage to the whole citrus industry.5,6
Additionally, Pseudomonas syringae pv. actinidiae (Psa) is the causal agent of
bacterial canker in kiwifruit and does huge damage to the international kiwifruit
production.7,8 On the other hand, it is well-known to us that fungal disease of the
crops is a major threat to global agricultural production all the time, from the
viewpoint of plant protection.9 For example, the fungus of Gibberella zeae is capable
of infecting a wide range of crop plants (including wheat and barley), and causing
head blight and rot diseases around the world.10,11 Although some agricultural
antimicrobial agents (like bismerthiazol, thiodiazole-copper, copper quinolate, zinc
thiazole, hymexazol, carbendazim, and thiophanate-methyl et al.) are now available
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for fighting against the aforementioned pathogens, the ever-increasing resistances of
them to currently-utilized antimicrobial agents12,13 and their adverse environmental
impacts posed by the abuse of these agrochemicals make it very urgent for the
development of new agricultural antimicrobial agents with novel structures and
improved performances.
Heterocyclic compounds, as the essential fragment of numerous commercialized
drugs and pesticides, play a crucial role in the process of developing medical and
agricultural antibiotics. Among them, the 1,2,4-triazole derivatives exhibit unique
derivatization advantages since this backbone is capable of having the substituents not
only at the 3- and 5-position but also at the 4-position, over its 1,3,4-oxadiazole and
1,3,4-thiadiazole couterparts. The introduction of additional substituent to 4-position
of the 1,2,4-triazole ring can be utilized to optimize the physicochemical features and
modulate the pharmacodynamics of target molecules.14 In particular, the
1,2,4-triazole-5(4H)-thione Mannich base derivatives were found to exhibit a wide
variety of bioactivities, including antibacterial,15 antifungal,16,17 anticancer,18
antitubercular,19 anti-inflammatory,20 and antiprotozoal21 effects (Figure 1). On the
other hand, 6-fluoroquinazoline derivatives are being attracting more and more
attention from medicinal and agrochemical chemists owing to their various biological
activities, such as antibacterial,22 antifungal,23 anti-TMV (tobacco mosaic virus),24
anti-HBV (hepatitis B virus),25 anticancer,26 antiproliferative,27 and anticonvulsant28
effects (Figure 1). Numerous studies had shown that the presence of piperidinyl
linkage within small molecules was essential for the observed bioactivity, which is
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also witnessed by their high prevalence in both drug and pesticide molecules
(including dopamine antagonist Clebopride, narcotic analgesic Fentanyl, antiallergic
drug Astemizole, and agrofungicide Oxathiapiprolin). Very interestingly, a statistical
analysis conducted by Njardarson and co-workers showed that the piperidine
heterocycle was the most prevalent nitrogen ring system among 640 drugs bearing a
nitrogen heterocycle (approved by FDA before the year 2014),29 followed by pyridine
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and piperazine rings, respectively.
Insert Figure 1
In our previous work,30 we synthesized a small group of 1,2,4-triazole-5-thione
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Mannich base-quinazolinone hybrids (compound I, Figure 2) and evaluated their
antibacterial and antifungal activities in agriculture. Unfortunately, the most potent
compound in this series was found to possess only moderate bactericidal efficacy
(~50% inhibiton rate) against Xoo at 100 μg/mL. After that, our group reported the
antimicrobial capabilities of quinazolinylpiperidinyl-containing 1,2,4-triazole
thioether acetamide derivatives (compound II).12 Delightedly, nearly all the target
compounds (except for two of them) exhibited comparable or even higher
antibacterial potencies against Xoo (the best compound inhibited Xoo with EC50 =
34.5 μg/mL in vitro) after comparison with control agent bismerthiazol. Very recently,
we prepared a series of 6-fluoroquinazolinylpiperidinyl-bearing 1,3,4-oxadiazole
thioether derivatives (compound III) and tested their antibacterial & antifungal
activities in vitro.22 Notably, four of them demonstrated significantly better
bactericidal activities against Xoo (the best compound suppressed Xoo with EC50 =
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26.0 μg/mL in vitro) than control bismerthiazol. However, the search of
easy-to-prepare, tunable molecular motifs that allow comprehensive structure-activity
relationship studies with the purpose of improving the bioactivity is crucial to develop
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new agrochemicals in the future.
Insert Figure 2
Based on the above-mentioned considerations, herein we designed and synthesized
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a series of 6-fluoroquinazolinylpiperidinyl-bearing 1,2,4-triazole-5-thione Mannich
base derivatives by the molecular hybridization approach (favorable for exerting
synergistic effects on the bioactivity,31,32 Figure 3). All the target compounds were
assessed in detail for their in vitro antibacterial and antifungal efficacies, against three
types of phytopathogenic bacteria and six types of phytopathogenic fungi of great
agricultural significance. Moreover, in vivo anti-Xoo activity was further tested for
highly-active compound 4f, in order to evaluate its practical application for the
prevention and control of rice bacterial blight. Finally, anti-Xoo mechanism of this
compound was preliminary explored using scanning electron microscopy (SEM)
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technique.
Insert Figure 3
118 2. Materials and Methods
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2.1. Instruments, Chemicals and Plant Pathogens
Melting points were determined on a XT-4 binocular microscope (Beijing Tech
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Instrument Co., China) and uncorrected. H and C NMR data were collected on a
Bruker Avance III 400 MHz NMR spectrometer at 298 K using DMSO-d6 as a
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solvent and TMS as an internal standard, and chemical shift (δ) was expressed in parts
per million (ppm). The following abbreviations were used in expressing the
multiplicity: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad
peak. High resolution mass spectra (HRMS-ESI) were recorded on a Thermo
Scientific Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer. IR spectra
were recorded on a Thermo Scientific Nicolet iS50 FT-IR spectrometer. Elemental
analyses were determined by an Elementar Vario-III elemental analyzer. SEM images
were visualized and obtained using a Nova NanoSEM 450. The X-ray
crystallographic data were collected using a Bruker Smart Apex CCD area detector
diffractometer (Bruker, Germany) with Mo-Kα radiation. All the chemicals were
purchased from commercial suppliers and directly used without further purification
(unless stated otherwise).
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All the strains of bacteria and fungi tested were provided by the Laboratory of Plant
Disease Control at Guizhou University.
2.2. In Vitro Antibacterial Bioassay
In vitro antibacterial activities of target compounds 4a4j and 7a7j were determined
against three plant pathogenic bacteria (Xoo, Xac, and Psa), according to the classical
turbidimetric method.12,33,34 The tested compounds were prepared at two
concentrations of 100 and 50 μg/mL. Pure DMSO in sterile distilled water was used
as a blank control, and commercially available agrobactericides bismerthiazol (BMT)
and thiodiazole-copper (TDC) were used as positive control agents (100 and 50
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μg/mL). About 40 μL of solvent NB (3.0 g of beef extract, 5.0 g of peptone, 1.0 g of
yeast powder, 10.0 g of glucose, 1000 mL of distilled water, pH = 7.0−7.2) containing
the bacterium Xoo, Xac or Psa was added to the mixed solvent system including 4 mL
of solvent NB and 1 mL of 0.1% Tween-20 containing tested compound or
BMT/TDC. The above test tube was incubated at 28 ± 1 °C and continuously shaken
at 180 rpm for one to three days. The bacterial growth was monitored by measuring
the optical density at 595 nm (OD595), given by turbiditycorrected values = ODbacterium
−
ODno bacterium, I = (Ctur − Ttur)/Ctur × 100%. The Ctur represented the corrected turbidity
value of bacterial growth of untreated NB (blank control), and Ttur denoted the
corrected turbidity value of bacterial growth of tested compound-treated NB. The I
represented inhibition ratio of tested compound against the bacterium.
Subsequently, antibacterial potencies of target compounds 4a4j and 7a7j were
further measured against Xoo under five different concentrations (namely 100, 50, 25,
12.5, and 6.25 μg/mL) to obtain their EC50 values, which were statistically determined
by Probit analysis using the software package SPSS 17.0.
2.3. In vivo Bioassay against Rice Bacterial Leaf Blight
The curative and protection activities in potted plants of compound 4f against rice
bacterial blight were determined, based on the previously-reported methods with
slight modifications.34,35 Bismerthiazol (20% wettable powder) and thiodiazole copper
(20% suspending agent), commercialized agrobactericides for controlling rice
bacterial leaf blight, were bought from the market and used as positive control agents.
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The curative activity for weakening rice bacterial leaf blight was tested under the
controlled conditions in a vertical incubator. After sowing rice seeds of variety
“Fengyouxiangzhan” about 8~10 weeks, rice leaves were then inoculated with Xoo,
which was incubated at the logarithmic growth by use of sterilized scissors. One day
after the inoculation, the solution of compound 4f, BMT, and TDC at 200 µg/mL
were uniformly sprayed into rice leaves, respectively. Meanwhile, distilled water was
also uniformly sprayed as the negative control. Subsequently, all the inoculated rice
plants were placed in a plant growth incubator (28 °C and 90% RH). At 14 days after
spraying, the disease index of the inoculated rice leaves was calculated. Likewise, the
protective activity in potted plants for weakening rice bacterial leaf blight was also
determined under the controlled conditions. After sowing rice seeds of variety
“Fengyouxiangzhan” about 8~10 weeks, the solution of compound 4f, BMT and TDC
at 200 µg/mL were uniformly sprayed onto rice leaves until dripping down.
Meanwhile, distilled water was also uniformly sprayed into the negative control plants.
One day after the spraying, Xoo, which was incubated at the logarithmic growth, was
inoculated on rice leaves through sterilized scissors. All the inoculated rice plants
were placed in a plant growth incubator (28 °C and 90% RH). At 14 days after the
inoculation, the disease index of inoculated rice leaves was measured. The control
efficiencies I (%) for the curative and protection activities were calculated using the
following equation. In this equation, C is the disease index of the negative control and
T is the disease index of the treatment group.
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I (%) = (C－T)/C × 100%
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2.4. Scanning Electron Microscopy (SEM)36
In this assay, 1.50 mL of Xoo cell suspension within the logarithmic growth phase
was centrifuged and washed with PBS buffer (pH = 7.2) three times, then
re-suspended them with 1.5 mL PBS buffer. Subsequently, these Xoo cells were
incubated with compound 4f at two different concentrations of 50.0 and 100.0 μg/mL
for 8 h, respectively. Meanwhile, an equiamount of DMSO was also used as a blank
control. After that, these samples were washed three times with PBS buffer (pH = 7.2).
Next, these Xoo cells were immobilized using 2.5% glutaraldehyde solution for 8 h at
4 °C, followed by dehydration with graded ethanol solution and absolute tert-butanol,
respectively. Finally, the samples were freezing dried, coated with gold and visualized
using Nova NanoSEM 450.
2.5. In vitro Antifungal Bioassay
The mycelial growth rate method13,37 was employed to assess in vitro fungicidal
activities of target compounds against six phytopathogenic fungi, including
Gibberella zeae, Verticillium dahliae, Sclerotinia sclerotiorum, Gloeosporium
fructigenum, Cytospora mandshurica, and Colletotrichum gloeosporioides. A DMSO
solution of the tested compound was added into sterilized Petri dishes, which
contained about 10 mL molten potato dextrose agar (PDA). Next, a 4.0 mm-diameter
of mycelial plug was cut from the fungal colony and placed at the center of PDA plate
at 28 ± 1 °C for three to six days. Antifungal assays were performed in triplicate for
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each compound. In addition, pure DMSO and commercialized agrofungicide
hymexazol were also tested as negative and positive controls, respectively.
The inhibition ratio (I) of the tested compound was calculated using the following
formula:
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I = (C−T)/(C−0.4) × 100%
In the formula, the C represented the average mycelial diameter of negative
control, and T represented the average mycelial diameter of the tested
compound-treated PDA.
2.6. Preparation of Intermediates 2, 3, 5, and 6
2.6.1. Synthesis of Intermediate 2
Acylhydrazine 122 (500 mg, 1.73 mmol) and phenyl isothiocyanate (0.25 mL, 2.07
mmol) were added to EtOH (20 mL), and the above mixture was heated to reflux for 6
h. After cooling down to room temperature, the formed precipitate was filtered,
washed with ethanol three times and dried to give amidothiourea 2.
2-(1-(6-fluoroquinazolin-4-yl)piperidine-4-carbonyl)-N-phenylhydrazinecarbothioami
1
de (2). Yield: 90%, m.p. 187–188 °C. H NMR (400 MHz, DMSO-d6, ppm) δ: 9.98
(br, 1H), 9.58 (br, 2H), 8.64 (s, 1H), 7.92–7.88 (m, 1H), 7.76 (t, J = 8.0 Hz, 1H), 7.69
(d, J = 8.0 Hz, 1H), 7.45 (d, J = 8.0 Hz, 2H), 7.34 (t, J = 8.0 Hz, 2H), 7.17 (t, J = 8.0
Hz, 1H), 4.28 (d, J = 16.0 Hz, 2H), 3.21 (t, J = 12.0 Hz, 2H), 2.64–2.59 (m, 1H), 1.98
13
(d, J = 8.0 Hz, 2H), 1.86–1.78 (m, 2H); C NMR (100 MHz, DMSO-d6, ppm) δ:
4
1
4
180.8, 173.9, 163.8 (d, JC-F = 4.0 Hz), 158.5 (d, JC-F = 243.0 Hz), 153.3 (d, JC-F
=
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2.0 Hz), 148.4, 139.2, 131.0 (d, 3JC-F = 9.0 Hz), 130.0, 128.1, 126.0, 122.5 (d, 2JC-F
=
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2
25.0 Hz), 116.6 (d, JC-F = 8.0 Hz), 109.4 (d, JC-F = 23.0 Hz), 56.1, 48.8, 27.9.
ESI-HRMS m/z: [M + H]+ calcd for C21H22FN6OS: 425.1554; found: 425.1538.
2.6.2. Synthesis of Intermediate 3
Amidothiourea 2 (500 mg, 1.18 mmol) was added to an aqueous solution of 5%
potassium carbonate (15 mL), and the above mixture was heated to reflux for 6 h.
After cooling down to room temperature, the reaction system was neutralized to pH =
7.0 with a 10% HCl solution, and the resultant precipitate was filtered, washed with
water and dried to give the key 1,2,4-triazole-5-thione 3.
3-(1-(6-fluoroquinazolin-4-yl)piperidin-4-yl)-4-phenyl-1H-1,2,4-triazole-5(4H)-thione
(3). Yield: 94%, m.p. 162–164 °C. 1H NMR (400 MHz, DMSO-d6, ppm) δ: 13.82 (br,
1H), 8.60 (s, 1H), 7.89–7.86 (m, 1H), 7.73 (t, J = 8.0 Hz, 1H), 7.64–7.57 (m, 4H),
7.51–7.49 (m, 2H), 4.17 (d, J = 12.0 Hz, 2H), 3.15–3.09 (m, 2H), 2.87–2.81 (m, 1H),
4
1.88–1.81 (m, 4H); 13C NMR (100 MHz, DMSO-d6, ppm) δ: 167.9, 163.5 (d, JC-F
=
1
4
5.0 Hz), 158.5 (d, JC-F = 243.0 Hz), 154.5, 153.2 (d, JC-F = 2.0 Hz), 148.4, 133.8,
3
2
131.0 (d, JC-F = 9.0 Hz), 129.7, 129.6, 128.5, 122.4 (d, JC-F = 25.0 Hz), 116.5 (d,
3JC-F = 8.0 Hz), 109.3 (d, 2JC-F = 24.0 Hz), 48.4, 32.5, 28.8. ESI-HRMS m/z: [M + H]+
calcd for C21H20FN6S: 407.1449; found: 407.1446.
2.6.3. Synthesis of Intermediate 5
Amidothiourea 5 was synthesized in a similar manner to amidothiourea 2, using
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4-(trifluoromethyl)phenyl isothiocyanate in place of phenyl isothiocyanate.
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2-(1-(6-fluoroquinazolin-4-yl)piperidine-4-carbonyl)-N-(4-(trifluoromethyl)phenyl)hy
1
drazinecarbothioamide (5). Yield: 93%, m.p. 163–164 °C. H NMR (400 MHz,
DMSO-d6, ppm) δ: 10.05 (br, 1H), 9.87 (br, 2H), 8.65 (s, 1H), 7.92–7.89 (m, 1H),
7.79–7.74 (m, 3H), 7.72–7.68 (m, 3H), 4.29 (d, J = 12.0 Hz, 2H), 3.23 (t, J = 12.0 Hz,
2H), 2.72–2.58 (m, 1H), 1.99 (d, J = 8.0 Hz, 2H), 1.88–1.83 (m, 2H); 13C NMR (100
4
1
MHz, DMSO-d6, ppm) δ: 180.9, 174.0, 163.8 (d, JC-F = 5.0 Hz), 158.5 (d, JC-F
=
4
3
244.0 Hz), 153.3 (d, JC-F = 2.0 Hz), 148.4, 143.1, 131.0 (d, JC-F = 9.0 Hz), 128.4,
125.7 (q, 2JC-F = 32.0 Hz), 125.2 (q, 3JC-F = 4.0 Hz), 123.3 (q, 1JC-F = 271.0 Hz), 122.5
2
3
2
(d, JC-F = 25.0 Hz), 116.5 (d, JC-F = 8.0 Hz) 109.4 (d, JC-F = 23.0 Hz), 56.1, 48.8,
27.9. ESI-HRMS m/z: [M + H]+ calcd for C22H21F4N6OS: 493.1428; found: 493.1408.
2.6.4. Synthesis of Intermediate 6
Similar to the preparation of 1,2,4-triazole-5-thione 3.
3-(1-(6-fluoroquinazolin-4-yl)piperidin-4-yl)-4-(4-(trifluoromethyl)phenyl)-1H-1,2,4-t
1
riazole-5(4H)-thione (6). Yield: 95%, m.p. 155–156 °C. H NMR (400 MHz,
DMSO-d6, ppm) δ: 13.71 (br, 1H), 8.60 (s, 1H), 8.01 (d, J = 8.0 Hz, 2H), 7.89–7.86
(m, 1H), 7.80 (d, J = 4.0 Hz, 2H), 7.74 (t, J = 8.0 Hz, 1H), 7.63 (d, J = 8.0 Hz, 1H),
4.17 (d, J = 12.0 Hz, 2H), 3.19–3.13 (m, 2H), 2.90–2.84 (m, 1H), 1.87–1.83 (m, 4H);
4
13C NMR (100 MHz, DMSO-d6, ppm) δ: 167.8, 163.5 (d, JC-F = 4.0 Hz), 158.5 (d,
1JC-F = 244.0 Hz), 154.3, 153.2 (d, 4JC-F = 2.0 Hz), 148.4, 137.5, 131.0 (d, 3JC-F = 9.0
Hz), 129.9 (q, 2JC-F = 32.0 Hz), 129.8, 126.8 (q, 3JC-F = 4.0 Hz), 123.9 (q, 1JC-F = 272.0
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Hz), 122.4 (d, JC-F = 24.0 Hz), 116.5 (d, JC-F = 9.0 Hz), 109.3 (d, JC-F = 23.0 Hz),
48.3, 32.3, 28.8. ESI-HRMS m/z: [M + H]+ calcd for C22H19F4N6S: 475.1323; found:
475.1306.
2.7. General Procedures for the Synthesis of Target Compounds 4a4j and 7a7j
A mixture of 1,2,4-triazole-5-thiones 3 or 6 (1.0 mmol) and 37% formaldehyde
solution (1.5 mmol) in ethanol (15 mL) were firstly stirred at room temperature for
0.5 h, and the appropriate secondary amine (1.2 mmol) was then added and
continuously stirred for 6~12 h. After the filtration, the crude product was dried and
recrystallized with dichloromethane/petroleum ether (1.0%, v/v) to afford target
compounds 4a4j and 7a7j.
2.8. Crystal Structure of Compound 4h
Single crystals of compound 4h suitable for X-ray diffraction analysis were obtained
by slow evaporation of a mixed DMF/EtOH (5.0%, v/v) solution of 4h at room
temperature. The crystal structure (Figure 4) revealed that compound 4h formed a 1:1
complex with water molecule through a hydrogen bond, involving water OH and
1-position nitrogen atom of the quinazoline backbone. Crystal data for complex
4hH2O (CCDC 1936090): Colorless crystal, C28H31FN8OS·H2O, Mr = 564.68,
triclinic, space group P-1, a = 8.600(6) Å, b = 9.829(7) Å, c = 18.434(13) Å; α =
75.578(19)°, β = 87.105(18)°, γ = 64.757(16)°; V = 1362.1(17) Å3, T = 296 K, Z = 2,
Dc = 1.377 g/cm3, F(000) = 596.0, reflections collected/independent reflections =
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4789/2188, goodness-of-fit on F2 = 1.015, R = 0.0642, wR2 = 0.1447.
Insert Figure 4
301 3. Results and Discussion
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3.1. Synthesis
Target compounds 4a4j were prepared in a consecutive seven-step reaction (as
depicted in Scheme 1), starting from 2-amino-5-fluorobenzoic acid. Briefly,
acylhydrazine 1 was firstly reacted with phenyl isothiocyanate in refluxing ethanol to
generate amidothiourea 2 in 90% yield, which then underwent a ring-closure reaction
under basic conditions and followed by the neturalization to give
1,2,4-triazole-5-thione 3 in 94% yield. Next, intermediate 3, formaldehyde solution
along with various secondary amines in ethanol solution were reacted at room
temperature to afford target compounds 4a4j. Some previous studies have shown
that the prescence of a trifluoromethyl group within bioactive compounds was capable
of improving the bioactivity.18,38 Thus, we also synthesized another class of target
compounds 7a7j containing a 4-trifluoromethylphenyl group at the 4-position of the
1,2,4-triazole-5-thione ring for comparison. It should be noted that no time-consuming
and laborious column chromatographic separation was required throughout the entire
reaction process (only through simple filtration, washing or recrystallization steps)
and good to excellent yields for all the intermediates and target compounds were
obtained, which were particularly important for the preparation of agrochemicals.
Insert Scheme 1
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3.2. In Vitro Antibacterial Activity
Antibacterial activities of target compounds 4a4j and 7a7j against three bacteria
Xoo, Xac, and Psa were evaluated in vitro by means of the classical turbidimetric
method, using commercialized agrobactericides bismerthiazol (BMT) and thiodiazole
copper (TDC) as positive control agents. As summarized in Table 1, all the target
compounds (except for compounds 4d and 7d) did exhibit a significantly higher
bactericidal potency against Xoo under two tested concentrations, as compared to
control BMT. In particular, compounds 4a, 4c, 4e, 4f, 4h, 4j, 7c, 7e, 7i, and 7j were
found to possess the inhibition rates 100%, 100%, 99.5%, 100%, 96.2%, 100%, 100%,
96.6%, 96.1%, and 100% towards this bacterium at 100 μg/mL, respectively, which
were considerably superior to that of control BMT (56.3%). In sharp contrast to Xoo,
only compound 7f across the series displayed comparable inhibition efficacies against
Xac, to those of control BMT. In addition, nine of them (including compounds 4b, 4j,
7b, 7c, 7d, 7f, 7g, 7i, and 7j) demonstrated similar bactericidal efficacies (>55%)
agasint the bacterium Psa at 100 μg/mL, to those of control agents BMT (59.6%) and
TDC (52.6%). The above observations indicated that 1,2,4-triazole Mannich base
derivatives bearing a 6-fluoroquinazolinylpiperidinyl moiety had a good antibacterial
selectivity towards Xoo over Xac and Psa.
Insert Table 1
Inspired by the preliminary antibacterial results shown in Table 1, EC50 values
(effective concentration for 50% inhibition activity) of these compounds against the
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bacterium Xoo were further determined utilizing the serial dilution method. As shown
in Table 2, all the compounds were found to possess lower EC50 values than control
BMT. In particular, compounds 4a, 4b, 4c, 4e, 4f, 4j, 7c, 7e, 7i, and 7j had EC50
values of 28.6, 30.2, 23.6, 28.0, 18.8, 23.4, 27.1, 27.9, 28.5, and 24.3 μg/mL,
respectively, over 3-fold more potent than BMT (EC50 = 92.4 μg/mL). In other words,
half of this class of compounds demonstrated far better bactericidal capabilities than
commericialized bactericide BMT.
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Insert Table 2
3.3. StructureActivity Relationship (SAR) Analysis for Target Compounds
against Xoo
Initially, we tested inhibition activities of compounds 4b, 4c, and 4d against Xoo,
having the pyrrolidine, piperidine and diethylamine moieties, respectively. Clearly,
compound 4d with a conformationally-flexible acyclic diethylamine group displayed
the weakest bactericidal activity among these three compounds, with EC50 values of
30.2, 23.6, and 72.0 μg/mL for compounds 4b, 4c, and 4d, respectively. The same
finding also held true for compounds 7b, 7c, and 7d, which contained a 4-CF3-phenyl
substituent on the 1,2,4-triazole-5-thione ring. Thus, the subsequent structural
optimizations for preparing the final Mannich base derivatives were mainly limited to
conformationally-rigid cyclic secondary amines, including morpholine, pyrrolidine,
piperidine, and various N-substituted piperazine derivatives. Overall, the presence of a
strong electron-withdrawing -CF3 group was detrimental or at least unhelpful to the
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anti-Xoo activity for the final compounds, with the exception of compounds 4i and 7i
(EC50 = 49.1 vs. 28.5 μg/mL). As far as the heterocycles containing a single nitrogen
atom were concerned, Mannich bases with a piperidine ring were identified as the best
bactericides, followed by those having the morpholine and pyrrolidine rings (4c > 4a
 4b and 7c > 7a > 7b). As for piperazine-derived Mannich bases, the presence of
weak electron-donating groups (such as compounds 4e/-CH3, 4f/-CH2CH3, and 4j/-Ph)
on the piperazine ring proved to be superior to those bearing the electron-withdrawing
groups (like compounds 4h/-COCH3 and 4i/-Boc). However, the existence of
sterically-bulky and weakly electron-donating iso-propyl group did not contribute to
the improvement of anti-Xoo activity, as exemplified by compounds 4g and 7g.
Among the whole series of compounds, Mannich base 4f with a N-ethyl piperazine
moiety demonstrated the highest anti-Xoo activity with an EC50 value of 18.8 μg/mL,
nearly 5-fold more potent than control BMT.
Given the particular importance of physicochemical properties in the development
of new pesticides,31,34,39,40 five parameters (namely, molecular weight (MW),
octanol/water partition coefficient (cLogP), number of hydrogen bond acceptors
(HBA), number of hydrogen bond donors (HBD), and number of rotatable bonds
(ROTB)) for target compounds 4a−4j and 7a−7j were obtained from the softwares of
ChemBioOffice 2010 and/or Molinspiration Cheminformatics (see Table 2).
Generally speaking, a promising pesticide candidate should meet the following
requirements (also known as Tice’s criteria41): (a) a cLogP value between 0 and 5; (b)
a MW magnitude ranging from 150 to 500; (c) a HBA number between 1 and 8; (d)
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HBD number  2; (e) ROTB number 12. Obviously, the most potent compound 4f
(having a relatively hydrophilic nature of cLogP = 3.17 relative to other compounds)
satisfied all the above listed requirements, except for molecular weight descriptor
(MW = 532.3, slightly larger than upper limit of 500 required by Tice’s criteria). On
the basis of these physicochemical features, this compound thus possessed a good
pesticide-likeness suitable for further studies.
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3.4. In Vivo Bioassay against Rice Bacterial Leaf Blight
In order to examine the practical potential of highly-active compound 4f, in vivo
bioassays against rice bacterial leaf blight were also carried out. As displayed in Table
3 and Figure 5, the obtained results revealed that compound 4f possessed an excellent
protection activity against Xoo at 200 μg/mL (with a control efficiency of 56.23%),
better than those of BMT (43.45%) and TDC (45.34%). Furthermore, compound 4f
was also found to have an outstanding curative activity against Xoo at 200 μg/mL
(with a control effect of 52.30%), higher than those of BMT (46.80%) and TDC
(42.43%). These findings implied that compound 4f had a good application
perspective as an alternative bactercide for the prevention and control of Xoo.
Insert Table 3
Insert Figure 5
3.5. SEM Studies on Cell Morphology
In order to explore the possible antibacterial mechanism, the morphological
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modifications of the bacterium Xoo in the presence of highly-active compound 4f
were monitored through the SEM technique. As seen in Figure 6a, the untreated
bacteria showed the form of elongated cells, having a firm surface and a well-shaped
appearance. After treatment with 50 μg/mL of compound 4f for 8 h, the appearance
was altered significantly. The size of Xoo was considerably reduced, and surface
collapses (accompanied by lots of pore-like structures) on the cell membranes were
clearly observed (Figure 6b). As the concentration rose to 100 μg/mL, more
accentuated morphological changes and few intact cells were simultaneously observed
(Figure 6c). These observations implied that compound 4f changed the permeability
of cell membranes, possibly leading to the leakage of the bacterial contents to the
extracellular environment.
Insert Figure 6
3.6. In Vitro Antifungal Activity
Antifungal activities of target compounds 4a−4j and 7a−7j were also assessed in vitro
against six types of fungal strains, according to the mycelial growth rate method. As
summarized in Table 4, most of the target compounds failed to exhibit notable
fungicidal activities at 50 μg/mL. However, four compounds (7d, 7e, 7g, and 7i)
revealed comparable antifungal efficiencies agaist G. zeae to control hymexazol
(55.0%), having the inhibition rates of 46.2%, 45.6%, 45.0%, and 45.6%, respectively.
In addition, compound 7i effectively suppressed the growth of V. dahlia at 50 μg/mL,
with the inhibition rate of 51.3%.
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Insert Table 4
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In summary,
a
series of easy-to-make and structurally tunable
6-fluoroquinazolinylpiperidinyl-containing 1,2,4-triazole Mannich base derivatives
were synthesized and assessed as antimicrobial agents in agriculture. Among them,
the structure of compound 4h was unambiguously confirmed by single crystal X-ray
diffraction analysis. The bioassays indicated that nearly all the target compounds
exhibited better inhibition activites against Xoo in vitro, relative to control
bismerthiazol. In particular, compounds 4c, 4f, 4j, and 7j possessed EC50 values
ranging from 18.8 to 24.3 μg/mL, considerably superior to that of commercialized
bactericide bismerthiazol (92.4 μg/mL). Moreover, the best compound 4f
demonstrated excellent protective and curative activities in vivo against rice bacterial
leaf blight caused by Xoo. The SEM measurements revealed that compound 4f
significantly changed bacterial cellular morphologies after interaction with Xoo.
Taken
together,
the
present
work
showed
the
potential
of
6-fluoroquinazolinylpiperidinyl-containing 1,2,4-triazole Mannich base derivatives as
a promising molecular platform for developing effective bactericides to control Xoo.
Acknowledgements
This work was financially supported by Science and Technology Program of Guizhou
Province (No. [2020]1Z025), Guizhou Provincial High-Level Overseas Talents
Innovation and Enterpreneurship Program (No. 201809), and Young Top-Notch
Talent Support Program of Guizhou Provincial Education Department (No. 2018038).
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448 Supporting Information
449
450
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452
Characterization data and original spectral files for intermediates 2, 3, 5 & 6 as well as
1
target compounds 4a4j & 7a7j (including H & 13C NMR) are available free of
charge on the ACS Publications website.
453 Conflict of interest
454
The authors declare no competing financial interest.
455 References
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