Synthesis, Crystal Structure, and Agricultural Antimicrobial Evaluation of Novel Quinazoline Thioether Derivatives Incorporating the 1,2,4-Triazolo[4,3- a]pyridine Moiety

Article abstract of DOI:10.1021/acs.jafc.9b04733

A total of 22 quinazoline thioether derivatives incorporating a 1,2,4-triazolo[4,3-a]pyridine moiety were designed, synthesized, and evaluated as antimicrobial agents in agriculture. Among these compounds, the chemical structure of compound 6l was further confirmed via single-crystal X-ray diffraction analysis. The bioassay results revealed that some of the compounds possessed noticeable in vitro antibacterial activities against the tested phytopathogenic bacteria. For example, compounds 6b and 6g had EC₅₀ values as low as 10.0 and 24.7 μg/mL against Xanthomonas axonopodis pv. citri (Xac), respectively, which were significantly better than that of the commercial agrobactericide bismerthiazol (56.9 μg/mL). Particularly, compound 6b was also found to be capable of suppressing the pathogenic bacterium Xanthomonas oryzae pv. oryzae (Xoo) approximately 12-fold more potent than control bismerthiazol, in terms of their EC₅₀ values (7.2 versus 89.8 μg/mL). Importantly, the most active compound 6b turned out to be one with the highest hydrophilicity and the lowest molecular weight within the series. In vivo bioassays further showed the application prospect of 6b as a promising plant bactericide for controlling Xoo. Additionally, in vitro antifungal activities of these compounds were also evaluated at the concentration of 50 μg/mL. Overall, the present study demonstrated the potential of 1,2,4-triazolo[4,3-a]pyridine-bearing quinazoline thioether derivatives as efficient agricultural antibacterial agents for crop protection.

Full text of DOI:10.1021/acs.jafc.9b04733

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Agricultural and Environmental Chemistry
Synthesis, Crystal Structure, and Agricultural Antimicrobial
Evaluation of Novel Quinazoline Thioether Derivatives
Incorporating the 1,2,4-Triazolo[4,3-a]pyridine Moiety
Zhijiang Fan, Jun Shi, Na Luo, Muhan Ding, and Xiaoping Bao
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b04733 • Publication Date (Web): 27 Sep 2019
Downloaded from pubs.acs.org on September 28, 2019
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Synthesis, Crystal Structure, and Agricultural Antimicrobial
Evaluation of Novel Quinazoline Thioether Derivatives
Incorporating the 1,2,4-Triazolo[4,3-a]pyridine Moiety
Zhijiang Fan, Jun Shi, Na Luo, Muhan Ding and Xiaoping Bao*
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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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These authors contributed equally to this work.
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Abstract
A total of twenty-two quinazoline thioether derivatives incorporating a
1,2,4-triazolo[4,3-a]pyridine moiety were designed, synthesized and evaluated as
antimicrobial agents in agriculture. Among these compounds, chemical structure of
compound 6l was further confirmed via single-crystal X-ray diffraction analysis. The
bioassay results revealed that some of the compounds possessed noticeable in vitro
antibacterial activities against the tested phytopathogenic bacteria. For example,
compounds 6b and 6g had EC50 values as low as 10.0 and 24.7 µg/mL against
Xanthomonas axonopodis pv. citri (Xac), respectively, which were significantly better
than that of commercial agrobactericide Bismerthiazol (56.9 µg/mL). Particularly,
compound 6b was also found to be capable of suppressing the pathogenic bacterium
Xanthomonas oryzae pv. oryzae (Xoo) approximately 12-fold more potent than
control Bismerthiazol, in terms of their EC50 values (7.2 versus 89.8 µg/mL).
Importantly, the most active compound 6b turned out to be one with the highest
hydrophilicity and the lowest molecular weight within the series. In vivo bioassays
further showed the application prospect of 6b as a promising plant bactericide for
controlling Xoo. Additionally, in vitro antifungal activities of these compounds were
also evaluated at the concentration of 50 µg/mL. Overall, the present study
demonstrated the potential of 1,2,4-triazolo[4,3-a]pyridine-bearing quinazoline
thioether derivatives as efficient agricultural antibacterial agents for crop protection.
Keywords
quinazoline, thioether, triazolo[4,3-a]pyridine, antimicrobial activity, SAR, synthesis
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1. Introduction
The phytopathogenic bacteria cause enormous economic losses in the world’s
agricultural production each year, especially Xanthomonas axonopodis pv. citri (Xac),
Xanthomonas oryzae pv. oryzae (Xoo) and Ralstonia solanacearum (Rs).1,2 Taking
Xoo as an example, it gives rise to rice bacterial blight via the invasion of vascular
system and xylem tissues from wounds or natural openings.1 Bacterial blight of rice is
one of the most deadly rice diseases across the world, which had been reported to
significantly affect rice yield with an estimate of a loss of nearly 50% in some Asian
countries.3 In addition, it is well-known that phytopathogenic fungi are the most
important crop diseases of all time,4 from the perspective of crop protection. Although
some agricultural antimicrobial agents are currently available from the market for
fighting against these pathogens, the search for new, more efficient and
environmentally-friendly antimicrobial agents remains a quite urgent task facing
agricultural scientists, especially considering an increasing resistance of plant
pathogens to currently-used antimicrobial agents in agriculture.5,6
The quinazoline backbone, which is composed of two fused six-membered
aromatic rings (namely a benzene ring and a pyrimidine ring), plays crucial roles in
the development and innovation of new drugs due to its various pharmacological
properties, such as antibacterial,79 antimalarial,10 anti-inflammatory,11 and
anticancer12 activities. Moreover, quinazoline derivatives also exhibited a wide variety
of pesticidal properties, including anti-phytobacterial,13,14 anti-TMV (tobacco mosaic
virus),15 insecticidal,16 and anti-phytofungal activities.1719 Some of quinazoline
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derivatives have been introduced into the market as drugs or pesticides, such as
anticancer drugs Gefitinib and Erlotinib, acaricide Fenazaquin, sympatholytic drug
Prazosin along with antihypertensive drug Doxazosin (Figure 1). On the other hand,
the 1,2,4-triazolo[4,3-a]pyridine scaffold is featured by a 1,2,4-triazole ring fused to a
pyridine ring and regarded as a privileged building block for the discovery of
drug/pesticide leads owing to their various bioactivities, like antibacterial,20
antifungal,21 anti-tumor,22 and herbicidal activities.23
The thioether linkage within bioactive molecules was believed to improve the
druglikeness, through decreasing the lipophilicity and functioning as a favorable
hydrogen-bonding acceptor.24,14 In particular, the diaryl thioether linkage was often
observed within a number of bioactive molecules as natural products, drugs or
pesticides,25,26 like anticancer drugs Axitinib and Thymitaq as well as herbicides
Pyrithiobac-sodium and Pyriftalid. In addition, the chemical modification of
halogenation is often utilized in the modern medicinal chemistry for improving the
potency and ADME (absorption, distribution, metabolism and excretion) properties of
drug molecules.27,28 Very recently, medicinal chemists have come to realize that the
formation of halogen-bonding interactions between the positive σ-hole of
halogen-substituted ligand and negatively-charged protein fractions actually
contributed to the stabilization of resultant ligand-protein complex.29 Interestingly,
previous studies had found that the chlorine substitution (-Cl) played a pivotal role in
the conformational stability of vancomycin-type antibiotics and anti-tumor activity of
salino-sporamide derivatives.27 Additionally, the Cl- interaction was important for
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therapeutic targets and it could also enhance the reactivity of certain biosynthetic
halogenases during the process of generating halogenated natural products.28
Bearing the above considerations in our mind and in continuation of our
longstanding efforts on developing new quinazoline derivatives as antimicrobial
agents in agriculture,6,13,14,17,18,30,31 herein we designed and synthesized a series of
novel
quinazoline
thioether
derivatives
containing
the
8-chloro-1,2,4-triazolo[4,3-a]pyridine moiety (Figure 2) via a pharmacophore
hybridization strategy (capable of exerting the synergistic effects,32 overcoming the
resistance,33 decreasing the toxicity and optimizing the pharmacokinetic profiles),
evaluated their in vitro antibacterial/antifungal activities against several important
phytopathogens of agricultural interest, and conducted a detailed structure-activity
analysis (SAR) based on the results of antibacterial bioassay. Subsequently, an in vivo
experiment was also carried out to further show the application prospect of the
highly-active compound as an agricultural bactericide for fighting against the plant
bacterium Xoo. To the best of our knowledge, this work represented the first example
of thioether-bridged quinazoline-1,2,4-triazolo[4,3-a]pyridine hybrids as efficient
agricultural bactericides for crop protection.
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Insert Figure 1
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Insert Figure 2
109 2. Materials and methods
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2.1 Instruments and Chemicals
Melting points were measured on a XT-4 binocular microscope (Beijing Tech
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Instrument Co., China) and uncorrected. All the NMR data (1H and C NMR) were
determined on a JEOL-ECX 500 NMR spectrometer at room temperature using TMS
as an internal standard, and chemical shift (δ) was expressed in parts per million
(ppm). The following abbreviations were utilized in expressing multiplicity: s =
singlet, d = doublet, t = triplet, q = quartet, m = multiplet. High resolution mass
spectra (HRMS-ESI) were recorded on a Thermo Scientific Q Exactive Hybrid
Quadrupole-Orbitrap mass spectrometer. 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 used without further purification (unless stated otherwise).
2.2 In vitro antibacterial assay
Antibacterial activities of target compounds were determined against three pathogenic
bacteria (Xoo, Xac and Rs), using the turbidimetric method.34,6 The tested compounds
were prepared at two concentrations of 200 and 100 μg/mL. Pure DMSO in sterile
distilled water was utilized as blank control, and commercial bactericides
bismerthiazol (BMT) and thiodiazole-copper (TDC) were used as positive control
agents. 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, 1 L of distilled water, pH = 7.0−7.2) containing the
bacterium Xoo/Xac/Rs was added to the mixed solvent system including 4.0 mL of
solvent NB and 1.0 mL of 0.1% Tween-20 aqueous solution containing tested
compound or BMT. The above test tube was incubated at 30 ± 1 °C and continuously
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shaken at 180 rpm for 13 days. The bacterial growth was monitored by measuring
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the optical density at 600 nm (OD600), 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 represented the
corrected turbidity value of bacterial growth of compound-treated NB. The I
represented inhibition rate of test compound against the bacterium.
Next, antibacterial activities of target compounds were further assessed against
Xac/Xoo under five different concentrations in order to obtain their EC50 values,
which were statistically determined by Probit analysis using the software package
SPSS 17.0.
2.3 In vivo anti-Xoo assay
The curative and protection activities in potted plants of compound 6b against rice
bacterial leaf blight were tested, according to the previously reported methods with a
slight modification.35 Bismerthiazol (20% wettable powder) and thiodiazole copper
(20% suspending agent), commercial bactericides for controlling rice bacterial leaf
blight, were bought from the market and employed as positive controls. The curative
activity for weakening rice bacterial leaf blight was determined under the controlled
conditions in a vertical incubator. After sowing rice seeds of variety
“Fengyouxiangzhan” about 8 weeks, rice leaves were then inoculated with Xoo, which
was incubated at the logarithmic growth by use of sterilized scissors. One day
following the inoculation, a solution of compound 6b at 200 µg/mL was uniformly
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sprayed onto rice leaves. 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 determined. 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
weeks, a solution of compound 6b at 200 µg/mL was 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 the equation, C is the disease index of the
negative control and T is the disease index of the treatment group.
I (%) = (CT)/C  100
2.4 In vitro antifungal assay
Mycelial growth rate method was utilized to evaluate in vitro antifungal activities of
target compounds against six types of phytopathogenic fungi.5 A DMSO solution of
tested compound was added into sterilized Petri dishes, which contained about 10 mL
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molten potato dextrose agar (PDA). Subsequently, a 4 mm-diameter of mycelial plug
was cut from the fungal colony and placed at the center of PDA plate at 28 ± 1 °C for
4 days. Antifungal assays were conducted in triplicate for each compound.
Additionally, pure DMSO and commercial fungicide (Hymexazol) were also
employed as negative and positive control agents, respectively.
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The inhibition rate (I) of the tested compound was determined based on the
following formula:
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 tested compound-treated PDA.
2.5 General Procedures for the Synthesis of Intermediates 5a5v
The 4-chloroquinazoline intermediates 5a5v were obtained by reaction of the
corresponding 2-substituted quinazolin-4-ones 4a4v36,37 with refluxing SOCl2 in the
presence of a catalytic amount of DMF38 and used directly in the next step.
2.6 General Procedures for the Synthesis of Target Compounds 6a6v
A mixture of 8-chloro-1,2,4-triazolo[4,3-a]pyridine-3-thiol 339 (300 mg, 1.62 mmol),
K2CO3 (447 mg, 3.24 mmol) and intermediate 5 (1.62 mmol) in acetonitrile solution
(25 mL) was stirred at room temperature overnight. Then, the formed precipitate was
filtered, washed three times with water, dried and recrystallized from ethanol to afford
target compounds 6a6v in 5973% yield.
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4-((8-chloro-[1,2,4]triazolo[4,3-a]pyridin-3-yl)thio)-2-(2-methoxyphenyl)quinazoline
(6l): yield 60%; mp: 175–176 °C; 1H NMR (500 MHz, DMSO-d6): δ 8.56 (d, J = 5.0
Hz, 1H), 8.37 (d, J = 5.0 Hz, 1H), 8.14–8.10 (m, 1H), 8.07 (d, J = 5.0 Hz, 1H),
7.90–7.87 (m, 1H), 7.81 (d, J = 7.6 Hz, 1H), 7.36–7.33 (m, 1H), 7.21–7.20 (m, 1H),
7.11 (t, J = 7.5 Hz, 1H), 6.96 (d, J = 5.0 Hz, 1H), 6.85 (t, J = 7.5 Hz, 1H), 3.40 (s, 3H);
13C NMR (125 MHz, DMSO-d6): δ 166.8, 160.1, 158.0, 149.9, 149.6, 136.5, 135.8,
131.8, 131.6, 129.2, 129.1, 128.9, 127.6, 125.0, 124.3, 121.1, 120.7, 120.4, 115.3,
112.8, 55.8; ESI-HRMS m/z: [M + H]+ calcd for C21H15N5OClS: 420.0680; found:
420.0673.
2.7 Crystal structure of Target Compound 6l
The chemical structure of compound 6l was clearly confirmed by X-ray single-crystal
diffraction analysis (Figure 3), by slow evaporation of an acetonitrile solution of this
compound at room temperature. Crystal data for 6l: colorless crystal, C21H14ClN5OS,
Mr = 419.88, triclinic, space group P-1; a = 9.4190(16) Å, b = 9.8354(16) Å, c =
11.1982(19) Å; α = 94.812(4)°, β = 100.746(3)°, γ = 110.614(3)°, V = 941.3(3) Å3, T
= 293 K, Z = 2, Dc = 1.481 g/cm3, F(000) = 432.0, reflections collected/independent
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reflections = 4667/2979, Goodness-of-fit on F2 = 1.024, R1 = 0.0522, wR2 = 0.1227.
Insert Figure 3
220 3. Results and Discussion
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3.1 Synthesis
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Overall, the synthesis of target compounds 6a6v was fairly straightforward and
accomplished in five steps from the starting materials (Scheme 1). In particular, no
chromatographic separation was required throughout the entire reaction processes. In
brief, 8-chloro-1,2,4-triazolo[4,3-a]pyridine-3-thiol 3 was reacted with the appropriate
2-substituted-4-chloro-quinazoline intermediates 5a5v in CH3CN using anhydrous
K2CO3 as catalyst to generate final compounds 6a6v. All the target compounds were
fully characterized by 1H NMR, 13C NMR and HRMS spectroscopic data.
Insert Scheme 1
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3.2 In vitro antibacterial activity
Antibacterial activities of target compounds 6a6v against three phytopathogenic
bacteria (namely Xoo, Xac and Rs) were examined by the turbidimetric method, using
commercially-available bactericides Bismerthiazol (BMT) and Thiodiazole-copper
(TDC) as positive control agents. As listed in Table 1, more than half of the target
compounds demonstrated similar or higher antibacterial activity against Xac at 100
µg/mL, compared with control BMT. In particular, the two most potent compounds
6b and 6g in this series were found to possess an inhibition rate of nearly 100%,
which were far superior to that of control BMT (62.4%). In addition, approximately
one third of target compounds exhibited comparable or even better inhibition activity
against Xoo at the concentration of 100 µg/mL, relative to control BMT. Especially,
compounds 6b and 6g were found to have an impressive bactericidal potency of 100%
and 94.4% against Xoo, respectively, far better than control BMT (57.1%). In contrast
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to the tested bacteria Xoo and Xac, almost all the compounds failed to display notable
antibacterial activity against Rs at 100 µg/mL, with the exception of compounds 6b
and 6h (close to 50% inhibition rate). Taken all together, compound 6b was found to
be the most efficient and broad-spectrum bactericide in the series, especially for
tackling phytopathogenic bacteria Xoo and Xac.
Insert Table 1
Encouraged by the preliminary antibacterial screening results, EC50 values
(effective concentration for 50% inhibition activity) of this class of compounds
against Xac were further determined using the serial dilution method. As summarized
in Table 2, nearly half of the tested compounds exhibited comparable or even lower
EC50 values, after comparison with control BMT. Among them, compounds 6b (R =
CO2C2H5), 6g (R = CO2CH(CH3)C2H5), 6m (R = 3-OCH3-Ph), 6o (R = 2-F-Ph), 6p (R
= 3-F-Ph), 6q (R = 4-F-Ph) and 6r (R = 2-Cl-Ph) demonstrated more potent
antibacterial potency against Xac with EC50 values of 10.0, 24.7, 33.0, 39.9, 32.4, 46.1
and 47.2 μg/mL, respectively, relative to control BMT (EC50 = 56.9 μg/mL).
Moreover, the rest of target compounds also showed moderate to good antibacterial
activity towards this bacterium, excluding compounds 6a, 6h, 6k and 6s.
Insert Table 2
On the other hand, considering the outstanding bactericidal activities displayed by
some compounds against Xoo in Table 1, their EC50 values were also measured. As
listed in Table 3, compounds 6b, 6c, 6f, 6g, 6p, 6q and 6t had EC50 values of 7.2, 71.2,
70.4, 13.8, 56.4, 83.4 and 67.8 μg/mL, respectively, better than control agent BMT
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(EC50 = 89.8 μg/mL). It was particularly noteworthy that carboxylic acid ethyl
ester-functionalized compound 6b exhibited the most potent antibacterial activity
against the pathogen Xoo, approximately 12-fold more active than control BMT.
Insert Table 3
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3.3 Structure-activity relationship analysis of target compounds against Xac
A preliminary structure-activity relationship (SAR) analysis of target compounds
against Xac was carried out and some preliminary conclusions could be drawn as
follows: a) Overall, the presence of carboxylic acid ester group at the 2-position of the
quinazoline scaffold was beneficial to the antibacterial potency. The two most active
compounds 6b and 6g of this series (possessing EC50 values of 10.0 and 24.7 μg/mL,
respectively) were discovered, both containing the carboxylic acid ester group; b)
Concerning halogen-containing compounds, fluorine-containing compounds
(including 2-F-Ph, 3-F-Ph and 4-F-Ph) exhibited higher anti-Xac activities relative to
their chlorine- and bromine-containing counterparts, which may be related to the
improved lipophilicity, metabolic stability and bioavailability of these compounds
after the introduction of fluoro-substituent.40,41 Additionally, previous studies had also
proved that the simultaneous presence of fluoride and sulfur elements within a single
molecule was particularly favorable for drug development;42,43 c) A subgroup of
twelve compounds 6i6t were purposedly synthesized to examine the influence of
substituent position at the benzene ring on the antibacterial activity, including weak
electron-donating -CH3, strong electron-donating -OCH3, strong electron-withdrawing
-F and moderate electron-withdrawing -Cl groups. For these groups, 4-position
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substitution was generally detrimental to the bactericidal activity in comparison to 2-
and 3-positional isomers, except for -Cl group; d) For carboxylic acid ester-containing
compounds, the presence of branched and straight-chain alkyl esters also significantly
affected the antibacterial activity, such as sec-butyl ester (compound 6g, EC50 = 24.7
μg/mL) versus isobutyl ester (compound 6f, EC50 = 59.5 μg/mL) and n-butyl ester
(compound 6e, EC50 = 53.3 μg/mL); e) Among the series, unsubstituted
phenyl/4-CH3-Ph/3-Cl-Ph-containing compounds (namely compounds 6h, 6k and 6s,
respectively) and non-substituted derivative at the quinazoline 2-position (i.e.,
compound 6a) were found to display the weakest antibacterial activity against the
pathogen Xac. Previous studies have shown that some physicochemical properties
(including molecular weight, lipophilicity/membrane permeability and polar surface
area et al.) of pesticide-active molecules were especially important for the
pesticide-likeness.4446 Therefore, molecular weight (MW), calculated n-octanol-water
partition coefficient (CLogP) and topological polar surface area (TPSA) of this class
of compounds were extracted from the software ChemBioDraw (Table 2). Very
interestingly, a combined analysis of all the data from Tables 13 indicated that
compound 6b was the best bactericide against three tested phytopathogenic bacteria,
which had the highest hydrophilicity, the lowest molecular weight (excluding
compound 6a, having no substituent at the quinazoline 2-position) and the largest
TPSA value. These findings suggested that more efficient bactericides were likely to
be discovered based on the quinazoline-thioether-1,2,4-triazolo[4,3-a]pyridine
molecular backbone, if further structural optimizations were conducted with the
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310
help of the fine modulation of the above physicochemical properties.
3.4 In vivo antibacterial activity
311
312
313
314
315
316
317
318
319
320
In order to assess the application prospect of highly-active compounds as agricultural
bactericides against phytobacterial disease, in vivo bioassays against rice bacterial leaf
blight were also conducted. As shown in Table 4 and Figure 4, the most active
compound 6b demonstrated good in vivo curative and protection activities against rice
bacterial leaf blight with the corresponding control efficiency of 48.54% and 49.47%
at 200 μg/mL, respectively, which were superior to BMT (40.63% and 38.82%,
respectively) and TDC (42.82% and 46.80%, respectively).
Insert Table 4
Insert Figure 4
321
322
3.5 In vitro antifungal activity
323
324
325
326
327
328
Antifungal activities of compounds 6a6v against six types of phytopathogenic fungi
were also evaluated in vitro at 50 μg/mL, using the mycelial growth rate method. As
shown in Table 5, almost all the compounds failed to exhibit noticeable fungicidal
activity (45%) against tested fungi, except for compound 6r against the fungi
Fusarium oxysporum and Verticillium dahliae (having the inhibition rates of 52.5%
and 65.4%, respectively), along with compound 6b against the pathogen Verticillium
329
330
331
dahliae (46.8%).
Insert Table 5
In summary, a series of novel quinazoline-thioether-1,2,4-triazolo[4,3-a]pyridine
332
derivatives were designed, synthesized and evaluated as antimicrobial agents in
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333
334
335
336
337
338
339
340
341
342
343
agriculture. The in vitro antibacterial assay showed that some compounds possessed
significantly better bactericidal activity, than commercial bactericide Bismerthiazol.
In particular, compound 6b containing a carboxylic acid ethyl ester group had EC50
values as low as 7.2 and 10.0 µg/mL against Xoo and Xac, respectively, significantly
better than commercial bactericide Bismerthiazol. Considering the relatively simple
molecular structure, ease of synthesis and workup (without any chromatographic
separation), and highly efficient antibacterial efficiencies of this class of target
compounds,
the
backbone
of
thioether-bridged
quinazoline-1,2,4-triazolo[4,3-a]pyridine could be considered as a promising
molecular platform for developing more efficient agrobactericides in the future.
344 Acknowledgement
345
346
347
348
This work was financially supported by Guizhou Provincial High-Level Overseas
Talents Innovation and Enterpreneurship Program (No. 201809), Young Top-Notch
Talent Support Program of Guizhou Provincial Education Department (No. 2018038)
and Breeding Program of Guizhou University (No. 20185781).
349 Supporting Information
350
351
352
1H NMR, 13C NMR, HRMS characterization data and original spectral files for target
compounds 6a6v (from Figure S1 to Figure S64) associated with this article can be
found in the online version at http://pubs.acs.org/journal/jafcau.
353 Conflict of interest
354
The authors declare no competing financial interest.
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355 References
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531 Figure Captions
532
Figure 1. Chemical structures of several representative bioactive molecules
containing quinazoline backbone or 1,2,4-triazolo[4,3-a]pyridine moiety
or thioether linkage.
533
534
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537
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541
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Figure 2. Design strategy of target compounds in this study.
Figure 3. X-ray crystal structure of target compound 6l.
Figure 4. Curative and protection activities of compound 6b against rice bacterial leaf
blight under greenhouse conditions at 200 μg/mL, with Bismerthiazol and
Thiodiazole-copper as positive control agents under the same conditions.
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560
Tables
561
562
Table 1. Antibacterial activities of target compounds 6a6v against three phytopathogenic
bacteria Xoo, Xac and Rs in vitro.
Inhibition ratea (%)
Xoo
100 µg/mL
Xac
100 µg/mL
Rs
Compd.
200 µg/mL
200 µg/mL
200 µg/mL
100 µg/mL
6a
6b
6c
6d
6e
6f
49.4 ± 3.7
100.0 ± 0.9
74.4 ± 1.9
66.3 ± 3.2
59.7 ± 1.2
70.2 ± 2.1
100.0 ± 0.2
62.5 ± 5.4
39.0 ± 3.6
36.7 ± 6.1
49.8 ± 4.6
31.2 ± 1.3
41.0 ± 2.5
64.7 ± 6.1
88.3 ± 3.1
81.8 ± 2.1
73.2 ± 2.5
61.2 ± 5.7
39.1 ± 1.9
75.6 ± 3.8
72.4 ± 3.1
38.6 ± 2.3
37.9 ± 1.3
100.0 ± 1.5
57.1 ± 2.3
44.7 ± 2.5
46.2 ± 1.6
58.6 ± 1.1
94.4 ± 2.4
43.5 ± 4.9
14.7 ± 4.3
19.2 ± 3.9
34.2 ± 5.1
28.3 ± 2.2
35.0 ± 0.8
34.9 ± 5.4
59.7 ± 0.8
63.1 ± 5.3
56.1 ± 1.6
25.5 ± 2.3
18.9 ± 3.0
64.0 ± 2.4
24.0 ± 2.1
21.3 ± 3.3
60.4 ± 3.0
100.0 ± 1.2
71.4 ± 2.6
73.1 ± 2.2
79.5 ± 1.9
72.3 ± 1.2
100.0 ± 1.2
54.1 ± 3.2
61.0 ± 2.5
66.7 ± 1.0
51.5 ± 2.8
73.4 ± 2.5
86.8 ± 3.4
81.3 ± 2.0
82.3 ± 2.6
83.4 ± 2.2
66.3 ± 1.5
76.9 ± 3.4
65.4 ± 3.7
75.9 ± 4.0
73.4 ± 5.1
77.9 ± 4.2
40.7 ± 3.1
100.0 ± 2.1
61.6 ± 1.7
57.2 ± 3.6
64.2 ± 2.5
58.3 ± 2.1
99.3 ± 1.1
45.6 ± 3.7
49.3 ± 3.4
55.3 ± 4.9
40.1 ± 3.1
58.3 ± 1.5
76.2 ± 2.2
51.5 ± 4.4
73.4 ± 2.3
74.4 ± 1.4
60.0 ± 3.1
66.1 ± 3.5
44.8 ± 6.8
68.5 ± 2.2
57.6 ± 5.5
63.6 ± 3.8
59.4 ± 1.6
56.1 ± 2.3
45.1 ± 3.3
51.1 ± 2.4
43.2 ± 1.5
51.2 ± 2.7
43.9 ± 2.7
51.4 ± 0.9
28.7 ± 2.8
46.8 ± 3.1
47.1 ± 1.3
35.7 ± 1.6
24.3 ± 1.0
28.7 ± 1.4
59.2 ± 3.1
46.8 ± 1.8
24.7 ± 1.1
57.2 ± 1.6
65.5 ± 1.3
29.5 ± 2.5
34.3 ± 0.8
49.0 ± 2.8
34.6 ± 2.5
46.4 ± 1.1
30.4 ± 2.3
29.3 ± 3.0
39.4 ± 2.1
36.4 ± 1.2
10.3 ± 0.8
46.0 ± 2.4
20.9 ± 1.2
30.2 ± 1.7
22.2 ± 1.5
21.1 ± 1.2
14.1 ± 2.1
14.9 ± 1.2
38.4 ± 1.8
39.7 ± 1.9
10.9 ± 0.8
30.1 ± 2.2
25.7 ± 1.9
14.1 ± 2.1
22.6 ± 1.1
27.8 ± 0.9
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
6s
6t
6u
6v
TDCb
BMTb
62.0 ± 1.8
79.0 ± 1.7
40.1 ± 3.1
57.1 ± 3.7
92.1 ± 3.0
83.6 ± 1.2
60.0 ± 1.2
62.4 ± 4.5
52.3 ± 4.8
NTc
21.2 ± 3.5
NTc
b
563 aThe average of three trials. Commercial bactericides Bismerthiazol (BMT) and Thiodiazole-copper (TDC) were
564 utilized as positive control agents. cNT = Not Tested.
565
566
567
568
569
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571
572
573
Table 2. EC50 values of target compounds 6a6v against Xac in vitro and their physicochemical
properties.
Toxic regression
Correlation
EC50
MWb
CLogPb
TPSAb
Compd.
equation
coefficient (r)
0.9944
0.9907
0.9984
0.9886
0.9978
0.9871
0.9926
0.9942
0.9781
0.9898
0.9916
0.9885
0.9966
0.9934
0.9915
0.9862
0.9845
0.9958
0.9991
0.9882
0.9934
0.9922
0.9965
(μg/mL)
139.0 ± 2.1
10.0 ± 1.8
58.7 ± 1.7
62.8 ± 5.2
53.3 ± 4.1
59.5 ± 2.1
24.7 ± 2.7
137.3 ± 3.7
91.8 ± 1.5
89.2 ± 3.2
166.9 ± 3.7
69.4 ± 4.1
33.0 ± 3.1
76.5 ± 4.3
39.9 ± 4.0
32.4 ± 1.6
46.1 ± 2.4
47.2 ± 3.4
121.8 ± 2.5
55.7 ± 1.8
83.1 ± 1.3
67.9 ± 2.8
56.9 ± 1.7
6a
6b
6c
y = 1.2661x + 2.2867
y = 2.0387x + 2.9595
y = 1.0661x + 3.1145
y = 1.0850x + 3.0491
y = 1.3621x + 2.6478
y = 0.9999x + 3.2260
y = 2.6850x + 1.2617
y = 1.0018x + 2.8585
y = 1.0846x + 2.8710
y = 1.2607x + 2.5412
y = 1.1702x + 2.3994
y = 1.2286x + 2.7380
y = 1.4725x + 2.7650
y = 1.8688x + 1.4803
y = 1.4307x + 2.7098
y = 1.2966x + 3.0416
y = 0.7236x + 3.7963
y = 1.2770x + 2.8626
y = 1.8058x + 1.2338
y = 1.6402x + 2.1359
y = 1.8125x + 1.5210
y = 1.9222x + 1.4791
y = 1.6307x + 2.1377
313.0
385.0
399.1
399.1
413.1
413.1
413.1
389.1
403.1
403.1
403.1
419.1
419.1
419.1
407.0
407.0
407.0
423.0
423.0
423.0
466.9
457.0
278.3
2.99
2.76
3.29
3.07
3.82
3.69
3.60
5.09
5.29
5.59
5.59
4.58
5.14
5.14
5.24
5.24
5.24
5.56
5.81
5.81
5.96
5.98
1.46
52.68
78.98
78.98
78.98
78.98
78.98
78.98
52.68
52.68
52.68
52.68
61.91
61.91
61.91
52.68
52.68
52.68
52.68
52.68
52.68
52.68
52.68
72.84
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
6s
6t
6u
6v
BMTa
b
574
575
aCommercial bactericide Bismerthiazol (BMT) was used as the positive control agent. These values were
extracted from ChemBioDraw Ultra 12.0.
576
577
578
579
580
581
582
583
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Journal of Agricultural and Food Chemistry
Page 28 of 37
584
585
586
587
588
589
590
Table 3. EC50 values of some compounds against Xoo in vitro.
Toxic regression
equation
Correlation
coefficient (r)
0.9792
EC50
Compd.
(μg/mL)
6b
6c
y = 3.5012x + 1.9999
y = 1.5325x + 2.1608
y = 1.0896x + 2.9871
y = 1.8634x + 2.8777
y = 1.9065x + 1.6605
y = 1.3295x + 2.4458
y = 1.3477x + 2.5318
y = 2.1405x + 0.8190
7.2 ± 1.5
71.2 ± 2.7
70.4 ± 1.2
13.8 ± 2.3
56.4 ± 3.1
83.4 ± 2.6
67.8 ± 1.4
89.8 ± 1.8
0.9936
6f
0.9911
6g
0.9948
6p
0.9913
6q
0.9897
6t
0.9799
BMTa
0.9908
591
592
aCommercial bactericide Bismerthiazol (BMT) was used as the positive control agent.
593
594
595
596
597
598
599
600
601
602
603
604
605
606
607
608
609
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Page 29 of 37
Journal of Agricultural and Food Chemistry
610
611
612
613
614
615
Table 4. Curative and protection activities of compound 6b against rice bacterial leaf blight under
greenhouse conditions at 200 μg/mL in vivo.
Protection activity (14 days after spraying)
Curative activity (14 days after spraying)
Treatmen
t
Morbidity Disease index Control efficiency Morbidity Disease index Control efficiency
(%)
100
100
100
100
(%)
(%)b
49.47
38.82
46.80
/
(%)
100
100
100
100
(%)
(%)b
48.54
40.63
42.82
/
6b
39.58
47.92
41.67
78.33
41.53
47.92
46.15
80.71
BMT
TDC
CKa
616
617
618
a Negative control. b Statistical analysis was conducted by ANOVA method under the condition of equal variances
assumed (P > 0.05) and equal variances not assumed (P < 0.05). Different uppercase letters indicate the values of
protection activity with significant difference among different treatment groups at P < 0.05.
619
620
621
622
623
624
625
626
627
628
629
630
631
632
633
634
635
636
29
ACS Paragon Plus Environment