Design, synthesis and antifungal evaluation of novel mandelic acid derivatives containing a 1,3,4-oxadiazothioether moiety

Article abstract of DOI:10.1111/cbdd.13861

A series of novel mandelic acid derivatives containing a 1,3,4-oxadiazothioether moiety were designed and synthesized. Bioassay results showed that some target compounds exhibited certain antifungal activity against six kinds of pathogenic fungi in vitro. Among the compounds, the EC₅₀ values of T₄₁ against Gibberella saubinetii, Verticillium dahlia and Sclerotinia sclerotiorum were 31.0, 27.0 and 32.1?μg/ml, respectively, and the EC₅₀ value of T₁₄ against S. sclerotiorum was 14.7?μg/ml. The antifungal activity against the resistant fungus S. sclerotiorum indicated that this series of target compounds may have the similar action modes or sites as the commercialized succinate dehydrogenase inhibitor carboxin. A morphological study with fluorescence microscope demonstrated that T₄₁ can significantly destroy the membrane integrity of G. saubinetii.

Full text of DOI:10.1111/cbdd.13861

Article type
: Research Article
Design, synthesis and antifungal evaluation of novel mandelic acid derivatives
containing a 1,3,4-oxadiazothioether moiety
Shuaitao Hou 1, Dewen Xie 2, Jingxin Yang 1, Xue Niu 3, Deyu Hu 1, *, Zhibing Wu 1, *
1 State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key Laboratory of Green Pesticide and
Agricultural Bioengineering, Ministry of Education, Center for R&D of Fine Chemicals of Guizhou University, Guiyang, 550025,
China.
2 School of Pharmaceutical Sciences Guizhou University, Guiyang, 550025, China.
3 School of Chemistry and Chemical Engineering, Guizhou University, Guiyang, 550025, China.
 Corresponding authors.
E-mail address: dyhu@gzu.edu.cn (D.Y. Hu); zbwu@gzu.edu.cn (Z.B. Wu).
Abstract
A series of novel mandelic acid derivatives containing a 1,3,4-oxadiazothioether moiety were designed and
synthesized. Bioassay results showed that some target compounds exhibited certain antifungal activity against
six kinds of pathogenic fungi in vitro. Among the compounds, the EC50 values of T41 against Gibberella
saubinetii, Verticillium dahlia and Sclerotinia sclerotiorum were 31.0, 27.0 and 32.1 μg/mL, respectively, and
the EC50 value of T14 against S. sclerotiorum was 14.7 μg/mL. The antifungal activity against the resistant
fungus S. sclerotiorum indicated that this series of target compounds may have the similar action modes or sites
as the commercialized succinate dehydrogenase inhibitor (SDHI) carboxin. A morphological study with
fluorescence microscope (FM) demonstrated that T41 can significantly destroy the membrane integrity of G.
saubinetii.
KEYWORDS
Mandelic acid derivatives, 1,3,4-oxadiazothioether, synthesis, antifungal activity, cross resistant
1 INTRODUCTION
Pathogenic fungi pose the greatest biotic challenge to plant health and thus to food security. However, the
emerging resistance of crop pathogens to fungicides poses a challenge to the discovery of new antifungal
compounds (Steinberg, 2020). This is of particular concern, as ~85% of currently used fungicides target single
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enzymes and can be overcome by single-point mutations. An alternative approach is the use of multi-site
fungicides, which usually interfere with unknown essential cellular processes in multiple ways (Leasbeater, &
Lucas, 2015). Currently, resistance development against succinate dehydrogenase inhibitors (SDHIs) and QoI
inhibitors (strobilurins) challenges agricultural security (Sparks, 2017). This provides an opportunity for us to
design novel active compounds with different action modes.
FIGURE 1 Reported compounds with 1,3,4-oxadiazole “thioether” or “sulfone” moiety derivatives.
In recent years, Song and Yang's research group has carried out systematic derivatization based on the
structures of 1,3,4-oxadiazole “thioether” and “sulfone” groups that exhibit a wide range of pesticidal activities,
including antibacterial (Li, 2013; Song, & Zheng, 2017; Li, 2018; Chen, & Shi, 2020), nematicidal (Li, 2017;
Chen, & Chen, 2018; Chen, 2020), antifungal (Xu, & Xu, 2011) and anti-TMV activities (Chen, 2016) (Figure
1). On the other hand, mandelic acid has very specific physiological activity, and is used as an important
intermediate to synthesize the medicines, such as cephalosporin antibiotics, vasodilators such as ring mandelic
acid, non-steroidal anti-inflammatory drugs such as norrecoxib and celecoxib, and urinary tract disinfectants
such as urotropine (Ding, 2018; Lukito, & Sajini 2019). In terms of pesticides, mandipropamid was the first
commercialized mandelic acid fungicide, marketed in 2001, and had a good control effect on
Pseudoperonospora cubensis and Pphytophora nicotianae (Keinath, & Qu, 2016).
To obtain compounds with high antifungal activity with novel action mechanisms and non-cross resistance
with commercialized SDHIs, a series of novel mandelic acid derivatives containing a 1,3,4-oxadiazothioether
moiety were designed and synthesized based on the previous work (Figure 2). In vitro antifungal bioassays
revealed that some target compounds exhibited certain antifungal activity against six kinds of pathogenic fungi.
Further antifungal activity against the resistant fungus S. sclerotiorum indicated that this series of target
compounds may have the same action mechanism as the commercialized fungicide carboxin. A morphological
study with a fluorescence microscope demonstrated that T41 can significantly disrupt the membrane integrity of
G. saubinetii.
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FIGURE 2 Design of the target compounds.
2 METHODS AND MATERIALS
2.1 Instruments and chemicals.
1H NMR and 13C NMR spectra were acquired on a Bruker 400 NMR spectrometer (Bruker Corporation,
Germany) with tetramethylsilane (TMS) as the internal standard and CDCl3 and DMSO-d6 as the solvent.
High-resolution mass spectrometry (HRMS) data were obtained on a Thermo Scientific Q Exactive (Thermo
Scientific, USA). The morphology of the fungus was observed by an Olympus-BX53F fluorescence microscope
(FM) (Olympus Ltd, Japan). Melting points were measured with an XT-4 binocular microscope melting point
apparatus (uncorrected). All reagents and solvents were of analytical grade.
2.2 Fungi.
Fungi (Gibberella saubinetii, Verticillium dahlia, Sclerotinia sclerotiorum, Alternaria longipes (Ellis & Everh.)
E.W. Mason, Alternaria solani and Diaporthe phaseolorum var. phaseolorum) were purchased from Beijing
Beina Chuanglian Biotechnology Institute, China. The resistant fungus Sclerotinia sclerotiorum was prepared
using the commercialized succinate dehydrogenase inhibitor (SDHI) carboxin with the reported literatures (Li,
2006; Chen, 2014; Zhang, 2014) in our laboratory in Guiyang City, China. These fungi were grown on potato
dextrose agar (PDA) plates at 25 ± 1 °C and maintained at 4 °C.
2.3 Synthesis. (Wu, 2016; Wang, 2013; Rosen, 2010; Song, 2005)
2.3.1 General synthetic procedure for intermediate 2
A mixture of DL-mandelic acid (50.0 g, 328.6 mmol), 98% sulfuric acid (16.1 g, 164.3 mmol) and methanol
(200 mL) was stirred at 80 °C for 2 h. Then, the solvent was concentrated in vacuo, and the residue was purified
by column chromatography on silica gel (petroleum ether/ethyl acetate 30/1) to obtain 2 as a white solid, yield
95%, m.p. 45-46 °C. 1H NMR (400 MHz, CDCl3) δ 7.45-7.35 (m, 5H, benzene H), 5.20 (d, J = 8.0 Hz, 1H, OH),
3.77 (s, 3H, OCH3), 3.56 (d, J = 8.0 Hz, 1H, CH); 13C NMR (101 MHz, CDCl3) δ 174.18, 138.25, 128.67,
128.56, 126.64, 72.91, 53.10.
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2.3.2 General synthesis procedure for intermediate 3
Intermediate 2 (51.8 g, 325.0 mmol) was dissolved in 80% hydrazine hydrate (50 mL), reacted at 100 °C for 30
min, cooled and filtered, and the filtrate was washed with water to obtain 3 as a white solid with a yield of 80%,
m.p. 118-119 °C. 1H NMR (400 MHz, DMSO-d6) δ: 9.18 (s, 1H, NH), 7.42-7.23 (m, 5H, benzene H), 5.99 (d, J
= 8.0 Hz, 1H, OH), 4.93 (d, J = 8.0 Hz, 1H, CH), 4.23 (s, 2H, NH2); 13C NMR (101 MHz, DMSO-d6) δ 171.52,
141.74, 128.35, 127.82, 126.94, 73.24.
2.3.3 General synthesis procedure for intermediate 4
To a solution of KOH (13.5 g, 240.7 mmol) in 200 mL of ethanol, 3 (20.0 g, 60.2 mmol) was added, and CS2
(13.7 g, 180.5 mmol) was slowly added dropwise into the solution and reacted at r.t. for 10 h and then reacted at
80 °C for 8 h. The solution was concentrated in vacuo. The pH value was adjusted to 6 with 2 M hydrochloric
acid solution, filtered, washed with water, dried and purified by column chromatography on silica gel (petroleum
ether/ethyl acetate 10/1) to obtain 4 as a light yellow oil, with a yield of 75%. 1H NMR (400 MHz, DMSO-d6) δ:
7.46-7.32 (m, 5H, benzene H), 6.79 (d, J = 4.0 Hz, 1H, OH), 5.84 (d, J = 2.0 Hz, 1H, CH).
2.3.4 General synthesis procedure for target compounds T11-T17, T21-T28, T31-T37 and T41-T47
To a solution of 4 (800 mg, 3.4 mmol) in CH3CN/H2O (5 mL/5 mL), KOH (929.2 mg, 6.7 mmol) and
substituted benzyl halide (483.6 mg, 3.5 mmol) were added and reacted at r.t. for 24 h and then concentrated in
vacuo. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate = 10/1 ~
20/1) to obtain the target compounds, and the physical and spectral data are provided in the supporting
information.
2.4 Bioassays.
The fungicidal activities of T11–T17, T21–T28, T31–T37 and T41–T47 were tested in vitro against six plant
pathogenic fungi (G. saubinetii, V. dahlia, S. sclerotiorum, A. longipes, A. solani, and D. phaseolorum) using a
mycelial growth inhibition method (Song, 2005; Yang, 2018). The preliminary activity screening concentration
of the target compounds was 100 μg/mL. The mycelial dishes of fungi that were used for testing were cut from
the PDA medium cultivated at 25 ± 1 °C and were approximately 4 mm in diameter, inoculated in the middle of
a PDA plate with a germ-free inoculation needle, and then were incubated for 4 to 5 days at the same
temperature. DMSO (1%) in sterile distilled water served as a blank control, whereas the commercialized
fungicides carboxin and mandipropamid were served as the positive controls. Each treatment was conducted in
triplicate. When the mycelia of the blank control grew to 6 cm, the diameter of the mycelia treated with the title
compounds was recorded. Inhibitory effects on these fungi were calculated by the formula I (%) = [(C − T)/(C −
0.4)] × 100, where C represents the diameter of fungal growth of the blank control, T represents the diameter of
the fungi with treated compound, and I represents the inhibition rate. Standard deviation (SD) values were
calculated based on the inhibition data of three repetitions for each test compound.
Based on the in vitro antifungal activity results, the median effective concentrations (EC50 values) of the
highly active compounds were further determined according to the method described above. A series of activity
screening concentrations of the target compounds and positive controls consisting of 200, 100, 50, 25, 12.5, 6.25,
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or 3.125 μg/mL were prepared. EC50 values were calculated with SPSS software 20 (Xu, 2011; Li, 2014). The
regression equations of the target compounds are provided in Tables S1 and S2 in the supporting information.
The fungicidal activities of some target compounds were also tested in vitro against the resistant fungus S.
sclerotiorum using the mycelial growth inhibition method described above.
2.5 Morphological observation of G. saubinetii under the T41 treatment
G. saubinetii was cultured in potato dextrose broth (PDB) medium at 25 °C and 180 rpm for 48 h and then
treated with 0, 1, and 2 eq. EC50 of compound T41 for 12 h. The PDB medium was removed by centrifuging at
4 °C and 6000 rpm for 5 min, and the hyphae were stained with 10 μL of a propidium iodide (PI) solution (20
μg/mL). The hyphae were incubated at 37 °C for 15 min in the dark and then washed with PBS three times. A
coverslip was placed on the hyphae, and the samples were observed and photographed using an
Olympus-BX53F FM (Zhang, & Zhang, 2018; Yang, & Zhang, 2020).
3 RESULTS AND DISCUSSION
3.1 Chemistry
The synthetic route of the target compounds T11–T17, T21–T28, T31–T37 and T41–T47 are depicted in Figure 3. As
an initial material, DL-mandelic acid was used to synthesize key intermediate ester 2. Then, 3 was obtained from
2 through hydrazinolysis in the presence of 80% hydrazine hydrate and reacted with CS2 under base conditions
to form intermediate thiol 4. Target compounds T11–T18, T21–T28, T31–T37 and T41–T47 were synthesized from 4
with different halogenated reagents or substituted benzyls with K2CO3 in acetonitrile (Wu, 2016; Wang, 2013;
1
Rosen, 2010). The target compounds were characterized by H nuclear magnetic resonance (NMR) and 13C
NMR spectroscopy and high-resolution mass spectrometry (HRMS).
FIGURE 3 Synthetic route of the target compounds.
3.2 Antifungal activity
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As indicated in Table 1, some target compounds exhibited certain antifungal activity against six kinds of plant
pathogenic fungi (G. saubinetii, V. dahlia, S. sclerotiorum, A. longipes, A. solani, and D. phaseolorum) at 100
μg/mL, such as T17, T27, T33, T34, T36, T37, T41, T44, T45 and T47. Among them, T41 and T46 exhibited 93.8% and
93.5% inhibition against V. dahlia, respectively, which were obviously superior than mandipropamid (27.2%).
Among these compounds, T41 exhibited good antifungal activities against three tested fungi (G. saubinetii, V.
dahlia, and S. sclerotiorum). As shown in Table 2, the EC50 values of T34 and T41 against G. saubinetii were
35.8 and 31.0 μg/mL, respectively. The EC50 values of T34, T41, T43 and T47 against V. dahlia were 38.5, 27.0,
30.0 and 33.5 μg/mL, respectively. The EC50 values of T13, T14, T25, T41 and T45 against S. sclerotiorum were
34.4, 14.7, 30.2, 32.1 and 39.7 μg/mL, respectively.
Preliminary structure–activity relationship (SAR) analysis revealed that the target compounds exhibited
improved antifungal activity when the chain length of the alkyl group on the thioether part increased, such as T14.
However, when the chain length of the alkyl was too long, the target compound lost its activity, such as that of
T15. The compounds exhibited better activities when CF3, CF3O, F, Cl or Br was introduced to the benzyl moiety.
However, compound T41 exhibited good antifungal activity against three plant phytogenic fungi because CH3
was introduced to the para-position of the benzyl group.
TABLE 1
Inhibition effect of target compounds against six plant phytopathogenic fungi at 100 μg/mL. a
Inhibition (%)
SS AL
compound
no.
GS
VD
AS
DP
T11
19.1±1.8
39.8±1.3
58.2±0.9
43.0±0.6
0
32.2±0.5
27.4±0.6
75.0±0.9
64.9±2.6
0
80.3±1.6
53.7±2.8
65.3±0.5
69.8±1.0
0
0
21.3±1.0
50.3±2.3
7.7±1.9
56.3±3.3
66.0±2.7
0
T12
0
0
T13
24.4±0.7
41.2±0.9
0
26.6±0.5
T14
43.4±2.0
T15
0
0
T16
28.3±2.7
36.6±0.9
55.1±1.3
24.8±2.4
14.1±0.6
T17
51.2±0.5
60.1±0.5
68.3±0.5
57.0±1.2
67.4±2.7
61.0±1.2
T21
T22
T23
T24
T25
T26
T27
T28
T31
T32
T33
T34
T35
37.9±0.5
38.2±1.8
39.7±0.5
59.7±0.9
51.3±1.1
36.2±0.6
73.4±0.6
58.7±0.8
30.8±2.6
38.0±0.5
57.7±1.0
76.0±1.1
42.5±1.2
64.9±3.0
69.3±0.5
30.8±0.5
67.9±0.9
54.1±0.5
49.7±2.2
73.7±5.3
70.5±0.5
24.3±0.8
48.7±0.5
52.9±0.5
84.5±5.7
58.3±1.4
47.8±0.5
44.9±2.6
65.0±1.3
59.0±0.8
65.8±1.0
52.4±0.7
62.2±0.4
64.4±0.4
43.3±0.8
50.3±1.3
59.1±0.4
59.1±0.4
64.9±0.5
47.4±1.2
52.8±0.9
47.8±3.3
57.9±0.5
31.2±0.8
44.1±1.0
56.3±2.0
49.5±1.7
44.1±1.0
33.8±1.2
50.5±1.0
66.7±1.3
57.6±1.4
59.8±1.7
58.2±0.5
58.8±0.5
55.0±0.5
18.6±1.0
56.0±0.8
66.5±1.0
59.3±0.9
27.5±2.7
35.3±0.7
59.0±1.1
77.4±2.4
64.3±1.8
63.1±1.7
26.6±2.0
24.4±1.5
24.0±1.9
47.0±2.2
67.5±3.6
73.5±4.0
68.1±4.7
41.0±2.4
63.0±2.8
66.6±2.9
64.9±1.6
75.9±4.0
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T36
65.4±0.5
66.7±0.5
68.8±0.8
40.8±1.2
52.9±1.3
55.5±0.8
51.2±0.5
64.8±1.6
59.6±0.5
19.1±0.4
35.2±1.9
80.4±1.8
77.4±2.3
93.8±0.9
38.9±0.5
47.2±0.7
69.9±1.4
59.8±0.5
93.5±4.4
77.4±3.1
27.2±0.9
100
66.1±0.5
66.1±1.3
70.7±1.4
36.6±1.4
43.7±0.5
55.8±0.5
65.9±0.5
62.4±1.0
64.7±0
58.8±1.6
51.4±1.4
50.8±1.2
41.2±4.3
22.2±2.0
51.8±2.1
59.5±0.9
57.6±1.4
57.2±1.3
12.6±1.3
47.9±2.4
68.6±1.1
65.9±1.3
57.5±2.7
35.5±0.5
49.7±0.5
69.5±3.0
64.3±1.8
65.6±1.2
65.6±1.5
10.7±0.5
87.6±6.8
69.6±3.1
70.8±4.5
74.7±3.9
14.4±2.5
67.5±4.0
73.2±3.7
78.0±3.9
0
T37
T41
T42
T43
T44
T45
T46
T47
72.6±3.3
34.5±1.8
0
mandipropamid
carboxin
14.0±0.5
88.1±6.4
GS: Gibberella saubinetii; VD: Verticillium dahlia; SS: Sclerotinia sclerotiorum; AL: Alternaria longipes (Ellis & Everh.) E.W.
Mason; AS: Alternaria solani; DP: Diaporthe phaseolorum var. phaseolorum.
a Values are means ± SD of three replicates.
TABLE 2
EC50 values of target compounds against three plant phytopathogenic fungi.a
EC50 (μg/mL)
No.
GS
VD
SS
＞200
＞200
T11
T12
T13
T14
T17
T21
T22
T23
T24
T25
T26
T27
T28
T32
T33
37.2±3.2
85.9±0.8
34.4±3.3
14.7±0.3
47.5±1.3
65.5±1.4
102.1±5.4
80.7±4.4
58.5±0.7
30.2±0.5
90.7±6.3
41.4±0.7
41.5±0.3
76.4±0.7
61.4±0.5
162.3±4.4
63.2±3.9
142.8±4.2
89.3±2.7
＞200
183.2±5.4
59.4±0.4
54.1±0.8
77.6±3.5
68.3±3.1
61.6±0.4
176.4±4.1
50.2.4±0.4
69.04±1.6
108.0±3.4
47.3±3.6
44.7±0.6
103.6±0.4
82.6±1.2
123.2±3.6
170.7±8.6
63.3±1.7
82.8±1.1
185.1±4.3
47.7±0.6
66.2±2.4
159.3±2.2
76.8±3.9
T34
T35
T36
T37
T41
T43
T44
T45
35.8±0.9
130.6±4.3
63.1±0.4
58.4±0.9
31.0±0.3
45.9±3.6
77.7±3.8
84.3±2.6
38.5±3.5
91.8±4.4
50.3±1.4
48.0±2.1
27.0±0.9
30.0±0.6
59.1±0.7
57.3±0.8
57.2±0.7
49.7±0.8
43.6±2.6
43.2±2.5
32.1±0.4
108.1±5.3
77.2±0.04
39.7±0.2
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T46
T47
57.5±2.7
65.1±3.2
42.9±1.3
33.5±5.6
47.7±1.8
48.9±0.9
GS: Gibberella saubinetii; VD: Verticillium dahlia; SS: Sclerotinia sclerotiorum.
a Values are means ± SD of three replicates.
The in vitro antifungal activity of the target compounds against the resistant fungus S. sclerotiorum was
also evaluated with a mycelial growth inhibition method (Song, 2005; Yang, 2018) at a concentration of 100
μg/mL. As shown in Table 3, the EC50 value of carboxin against the resistant S. sclerotiorum (243.8 μg/mL) was
nearly 20 times that against S. sclerotiorum (12.6 μg/mL), which indicated that this resistant fungus S.
sclerotiorum can be used for cross-resistance evaluation.
TABLE 3
EC50 values of carboxin against fungus. a
fungus
EC50 (μg/mL)
12.6±2.8
the toxic regression
y = 1.466x + 3.3871
y = 0.7468x + 3.2173
R²
S. sclerotiorum
0.922
0.930
resistant S. sclerotiorum
243.8±4.6
a Values are means ± SD of three replicates.
As shown in Table 4, the antifungal activity of the target compounds against the resistant S. sclerotiorum
obviously decreased compared to those against S. sclerotiorum, especially for compound T41. The inhibition rate
of T41 was decreased from 70.7% to 4.4% at a concentration of 100 μg/mL, which indicated that this series of
target compounds may have the similar action mechanism as the commercialized SDHI carboxin.
TABLE 4
Inhibition effect of target compounds against the resistant fungus S. sclerotiorum at 100 μg/mL. a
compound
no.
compound
no.
Inhibition (%)
Inhibition (%)
T11
25.6±0.4
24.6±5.0
44.6±2.3
47.2±2.5
T35
42.8±1.9
50.8±1.5
63.9±2.7
4.4±0.9
T13
T14
T17
T36
T37
T41
T23
T25
T27
48.0±2.5
51.3±0.4
47.0±6.1
T45
T46
T47
51.8±0.4
46.9±0.8
36.9±2.8
T28
59.5±0.4
carboxin
36.7±1.1
a Values are means ± SD of three replicates.
3.3 Effect of compound T41 on fungal morphology
As shown in panels A, B and C of Figure 4, the cellular membrane of the hyphal cells without staining was
colourless. PI can enter the cell across the damaged cellular membrane and specifically bind with DNA to emit
red fluorescence. Compared with the blank control (Figure 4D), a strong fluorescence intensity was observed in
the G. saubinetii hyphae treated with compound T41 at a concentration of 2 eq. EC50 and stained with PI (Figure
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4F). As shown in Figure 4E and 4F, the higher compound T41 concentration was, the more red hyphae were
present. Therefore, the FM observation further proved that target compound T41 could destroy the membrane
integrity of G. saubinetii, and the effect was dose dependent.
FIGURE 4 Morphology of G. saubinetii under optical microscopy and fluorescence microscopy.
(A, B and C: bright field; D, E and F: fluorescence field. A, D 0 μg/mL (CK); B, E: 1 eq. EC50; C, F: 2 eq. EC50)
4 CONCLUSIONS
In summary, twenty-nine novel mandelic acid derivatives containing a 1,3,4-oxadiazothioether moiety were
designed and synthesized. Bioassay results showed that some target compounds exhibited certain antifungal
activity against six kinds of pathogenic fungi in vitro. Among the target compounds, some exhibited good
antifungal activity against G. saubinetii, V. dahlia and S. sclerotiorum. The EC50 values of T41 against G.
saubinetii, V. dahlia and S. sclerotiorumwere 31.0, 27.0 and 32.1 μg/mL, respectively, and the EC50 value of T14
against S. sclerotiorum was 14.7 μg/mL. The antifungal activity against the resistant fungus S. sclerotiorum
indicated that this series of target compounds may have the similar action modes or sites as the commercialized
SDHI carboxin. A morphological study with FM demonstrated that T41 can significantly destroy the membrane
integrity of G. saubinetii. Subsequent mechanism research is ongoing.
FUNDING
This work was supported by the National Natural Science Foundation of China (No. 21662008), and the Science
and Technology Project of Guizhou Province (No. [2017]5788).
CONFLICTS OF INTEREST
The authors declare that there are no conflicts of interest regarding the publication of this article.
SUPPORTING INFORMATION
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Supporting information may be found in the online version of this article.
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