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Synthesis, bioactivity and preliminary mechanism of action of novel trifluoromethyl pyrimidine derivatives
⁎Corresponding authors. wuwenneng123@126.com (Wenneng Wu), wxue@gzu.edu.cn (Wei Xue)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Abstract
In this study, a series of trifluoromethyl pyrimidine derivatives 5a-5v were designed and synthesized. All synthetic compounds were original. Bioassay results showed that some of the target compounds were proved to have higher antiviral and antifungal activities than those of commercial agents. Especially, EC50 values of the curative activity of compound 5j and the protection activity of compound 5m were 126.4 and 103.4 µg/mL, respectively, which were lower than that of ningnanmycin. Microscale thermophoresis experiment proved that there was a good interaction between compound 5m and TMV-CP. Meanwhile, the antifungal activity results showed that compound 5u had a significant on in vitro against Rhizoctonia solani (RS) activity, with the EC50 value of 26.0 µg/mL, which was equal to that of azoxystrobin. As well, in vivo experiments on rice leaves showed that compound 5u could effectively control RS, and the effect of 5u on the cell morphology of RS was observed by scanning electron microscopy.
Keywords
Trifluoromethyl pyrimidine
Antiviral activity
Antifungal activity
Mechanism of action
- 1H NMR
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1H nuclear magnetic resonance
- 13C NMR
-
13C nuclear magnetic resonance
- 19F NMR
-
19F nuclear magnetic resonance
- HRMS
-
High-resolution mass spectrometry
- EC50
-
Median effective concentration
- TMV
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Tobacco mosaic virus
- AB
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Alternaria brassicae
- FF
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Fusarium fujikuroi
- FO
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Fusarium oxysporum f.sp.cucumerinum
- CT
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Colletotrichum truncatum
- PC
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Phytophthora capsici
- CG
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Colletotrichum gloeosporioides
- RS
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Rhizoctonia solani
- FG
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Fusarium graminearum
- PS
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Phytophthora sojae
- PP
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Phytophthora palmivora
- BC
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Botrytis cinerea
- PL
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Phytophthora litchii
Abbreviations
1 Introduction
Food is the basic need of mankind to maintain the health and survival, as well as an important strategic material that affects national security, political security and economic development (Hao et al., 2020). In our daily life, grains, vegetables, and fruits are important components of human food (Chen et al., 2018). However, plant diseases caused by bacteria, fungi, and viruses are extremely difficult to control in agricultural production, seriously affecting the yields and quality of crops and causing huge economic losses (Ji et al., 2016; Lu et al., 2014). Chemical agents are one of the important ways to protect crop products. Long-term use and irregular use inevitably lead to increasing drug resistance and prominent ecological risks (Zhang et al., 2019). Therefore, it is of great significance to develop new pesticides with new structures and high activity (Jiang et al., 2020).
Pyrimidine is an important part of N-containing heterocyclic compound that were widely distributed in the nature, such as caffeine, vitamin B, theophylline, and so on (Liu et al., 2021a,b). Meanwhile, due to its extensive pesticide biological activities, such as anticancer (Wang et al., 2021; Munikrishnappa et al., 2021; Alia et al., 2021; Sankarganesha et al., 2021; Zühal et al., 2020), anti-inflammatory (Purushothaman et al., 2018), antimalarial (Mane et al., 2014), anti-leukemia (Yang et al., 2013), antitumor (Liu et al., 2021a,b; Guo et al., 2020a,b), immunomodulatory (Dolsak et al., 2021), insecticide (Wu et al., 2019a,b; Chen et al., 2013), herbicidal (Zuo et al., 2016), antiviral (Zan et al., 2020; Mamdouh et al., 2021; Alaa et al., 2022) and antifungal (Zhang et al., 2016; Mohammareh et al., 2020; Chen et al., 2022; Samata et al., 2022) activity, etc (Wang et al., 2017; Seenaiah et al., 2014; Emami et al., 2020), pyrimidine was found to be one of the most important active fragments in pesticide chemistry and some commercialized pyrimidine pesticides (Fig. 1) have been successfully developed. The reason for introducing phenyl ether and ester moieties into the molecular skeleton, on the one hand, because these groups are found in many commercial drugs, and on the other hand, the introduction of these groups can increase the water solubility of compounds. In our previous work, we also found that pyrimidine derivatives revealed good antifungal (Wu et al., 2021), antiviral (Wu et al., 2015), insecticidal (Wu et al., 2020), and antibacterial activities (Su et al., 2021).
The structures of some commercialized pyrimidine pesticides.
In this study, considering that pyrimidine compounds had a wide range of biological activities, we obtained a series of trifluoromethyl pyrimidine derivatives through ring closure, chlorination, substitution and other reactions from ethyl trifluoroacetoacetate. The target compounds were screened for antiviral and antifungal activity. Subsequently, Microscale thermophoresis (MST) and molecular docking experiments were performed on compound 5m with excellent antiviral activity to further confirm its application prospects in the development of antiviral agents. In addition, in vivo experiment of 5u against Rhizoctonia solani (RS) was carried out and the effects of 5u on RS cells was observed and evaluated by scanning electron microscopy. The antifungal action mechanism of the target compound 5u was preliminarily discussed.
2 Materials and methods
2.1 Instruments and chemicals
The melting points were determined by using an XT-4 binocular microscope (Beijing Tech. Instrument Co., China) and uncorrected. Proton nuclear magnetic resonance 1H NMR, 13C NMR, and 19F NMR spectra were obtained by using an ASCEND 400 NMR (Bruker Optics, Switzerland) spectrometer operated at room temperature with CDCl3 as the solvent and tetramethylsilane as the internal standard. High-resolution mass spectrometry (HRMS) was conducted by using a Thermo Scientific Q Exactive (Thermo Scientific, Missouri, USA). The X-ray crystal data were obtained by Bruker D8-QUEST diffractometer (Bruker Optics, Switzerland). The chemical materials and reagents were purchased from commercial suppliers and all reagents are analytical pure solvents made in China.
2.2 Synthesis
General Procedures for Preparing Intermediates 1–4 and the Compounds 5a–5v. A series of trifluoromethyl pyrimidine derivatives (5a-5v) were designed and synthesized according to Scheme 1. Intermediates 1, 2, 3, and 4 were synthesized by the reported methods (Wu et al., 2019a,b; Yu et al., 2021). The intermediate 4, halohydrocarbon and K2CO3 were reacted in DMF for 6–8 h at ice-bath. TLC was used to monitor the reaction. The crude product was purified by column chromatography (V/V, petroleum ether: ethyl acetate = 15:1 to 8:1) to obtain the compounds 5a-5v. The specific preparation methods of the target compounds 5a-5v were provided in Supporting Information.
Synthetic route of the target compounds 5a-5v.
2.3 Antiviral bioactivity
The anti-TMV activities assay of target compounds according to the method had reported (Luo et al., 2020; Chen et al., 2020). The microscale thermophoresis and molecular docking have been performed using our previously reported methods (Guo et al., 2020a,b; Tang et al., 2019). Common leaf K326 (Nicotiana Tabacum K326), extracted from tobacco Mosaic virus; Nicotiana glutinosa, the host of TMV.
2.4 Antifungal bioactivity
The in vitro and in vivo antifungal assay, sclerotia germination inhibition sclerotia formation inhibition tests and scanning electron microscopy experiments were performed according to the literature methods reported (Wang et al., 2019; Zhang et al., 2018a,b; Shi et al., 2020; Zhang et al., 2018a,b).
3 Results and discussion
3.1 Chemistry
As shown in Scheme 1, compounds 5a-5v were synthesized, and characterized their structures by 1H NMR, 13C NMR, 19F NMR and HRMS. All synthetic compounds were original. The specific data were displayed in the Supporting Information.
Meanwhile in order to further determine the structure of the target compounds, compounds 5d and 5f were successfully cultured into a single crystal and analyzed their structures by single crystal X-ray diffraction. The crystal structures of 5d (deposition CCDC 2121760) and 5f (deposition CCDC 2121761) were shown in Fig. 2. More characterization data were supplied in the Supporting Information.
The crystal X-ray structures of compounds 5d (A) and 5f (B).
3.2 Antiviral assay
3.2.1 Antiviral activity of compounds 5a-5v against TMV in vivo
The half-leaf spot method was used to evaluate the antiviral activity of the target compounds against TMV in vivo at 500 µg/mL. Table 1 showed that the target compounds 5a-5v exhibited moderate to good anti-TMV activity, with the curative, protection, and inactivation activity ranges of 32.4–76.3%, 23.9–66.4%, and 23.2–76.7%, respectively. Among of them, compounds 5b, 5j, 5l, 5m, 5q, and 5s had significant curative activity against TMV, with the inhibition rates of 70.1, 76.3, 64.1, 66.1, 71.3 and 64.2%, respectively, which were better than that of ningnanmycin (54.0%). Meanwhile, compounds 5f, 5h, 5m, 5q, and 5s showed remarkable protection activity against TMV, with the inhibition rates of 65.7, 66.4, 65.5, 62.7 and 61.6%, respectively, which were superior to ningnanmycin (58.6%). In vivo leaf diagram of compound 5m was shown in Fig. 3.
Compounds
Curative activity (%)a
Protection activity (%)a
Inactivated activity (%)a
5a
59.8 ± 0.1
58.1 ± 0.2
49.6 ± 0.1
5b
70.1 ± 5.2
42.1 ± 3.7
41.4 ± 0.2
5c
44.4 ± 0.3
42.9 ± 4.5
45.1 ± 0.4
5d
32.8 ± 0.1
45.6 ± 2.5
56.1 ± 0.2
5e
48.2 ± 0.1
35.2 ± 0.1
76.7 ± 0.1
5f
55.6 ± 0.7
65.7 ± 0.2
56.9 ± 0.2
5g
49.1 ± 0.1
56.9 ± 5.0
45.2 ± 0.1
5h
38.0 ± 5.2
66.4 ± 0.2
51.5 ± 0.1
5i
39.3 ± 6.1
42.2 ± 3.8
64.1 ± 0.1
5j
76.3 ± 0.1
49.1 ± 1.5
51.4 ± 0.2
5k
56.0 ± 0.6
62.1 ± 0.8
23.2 ± 1.2
5l
64.1 ± 0.6
32.9 ± 0.5
64.0 ± 0.2
5m
66.1 ± 0.7
65.5 ± 0.3
56.4 ± 0.1
5n
48.7 ± 0.1
37.1 ± 0.6
46.5 ± 0.1
5o
42.7 ± 0.1
38.8 ± 2.5
46.1 ± 0.2
5p
54.4 ± 3.0
55.8 ± 1.0
57.5 ± 0.1
5q
71.3 ± 0.1
62.7 ± 1.0
51.1 ± 4.9
5r
32.4 ± 0.1
47.7 ± 0.1
69.3 ± 2.9
5s
64.2 ± 0.6
61.6 ± 0.1
69.3 ± 1.0
5t
53.1 ± 0.1
23.9 ± 0.9
66.9 ± 2.8
5u
41.1 ± 1.4
60.4 ± 0.7
39.7 ± 2.4
5v
50.8 ± 1.2
26.2 ± 2.5
60.6 ± 0.2
Ningnanmycinb
54.0 ± 0.2
58.6 ± 0.7
94.9 ± 0.1
Anti-TMV activities of 5m (A. Curative activity, B. Protection activity, C. Inactivated activity) and ningnanmycin (D. Curative activity, E. Protection activity, F. Inactivated activity) in vivo.
EC50 values of some target compounds were further evaluated and the test results were listed in Table 2. Table 2 showed that the tested compounds 5b, 5f, 5h, 5j, 5k, 5l, 5m, 5q and 5s revealed better curative or protective activity, and EC50 values were better than those of ningnanmycin. Among of them, compound 5j has the best curative activity, with EC50 value of 126.4 µg/mL, which was better than that of ningnanmycin (362.7 µg/mL). Compound 5m showed significant protective activity with EC50 value of 103.4 µg/mL, which was superior to that of ningnanmycin (255.1 µg/mL).
Compounds
EC50 (µg/mL)a
Curative activity
Protection activity
5b
205.4
–
5f
–
122.1
5h
–
111.6
5j
126.4
–
5k
–
191.8
5l
257.8
–
5m
169.1
103.4
5q
139.3
191.2
5s
174.9
–
Ningnanmycinb
362.7
255.1
The structure–activity relationship (SAR) analyzed on the basis of the antiviral activities against TMV shown in Table 1. When R1 and R2 was CH3 group, the target compound had the best protective activity against TMV. For example, the protective activities of compounds 5h (R1 = CH3, R2 = CH3), 5f (R1 = CH3, R2 = 2-Cl-6-F-Ph) and 5k (R1 = CH3, R2 = 2-Cl-5-thiazoly) were 66.4, 65.7 and 62.1%, respectively, which were superior to other substituents. In addition, when R1 was H, the protective activities of compounds 5m (R1 = H, R2 = 2-F-Ph), 5q (R1 = H, R2 = 2-Cl-6-F-Ph), 5s (R1 = H, R2 = CH3) and 5v (R1 = H, R2 = 2-Cl-5-thiazoly) were 65.5, 62.7, 61.6 and 60.4%, respectively. In terms of the curative activity data of the target compounds against TMV, compounds 5j (76.3%) > 5q (71.3%) > 5b (70.1%) > 5m (66.1%) > 5s (64.2%) > 5l (64.1%), in which compounds 5b (R1 = CH3, R2 = 2-F-Ph) and 5m (R1 = H, R2 = 2-F-Ph) had different R1 groups and same R2 groups. It is speculated that when 2-F-Ph at the R2 position, R1 was electron donor group which could enhance the protective and curative activities of the target compounds against TMV at 500 µg/mL. It can be seen from the test results of anti-TMV activity in Table 1 that compound 5s (R1 = H, R2 = CH3) has excellent anti-TMV activity in terms of protective, curative and inactivated activities compared with compounds 5h (R1 = CH3, R2 = CH3) and 5o (R1 = H, R2 = 2-Cl-Ph) of the same series. Indicating that the introduction of a CH3 group at the R2 position could improve the biological activity of the compound under the condition that the H atom at the 2-position of the pyrimidine remains unchanged.
3.2.2 Binding sites of 5h, 5m, 5t and ningnanmycin to TMV-CP
Microscale thermophoresis (MST) was used to further analyze the interaction of compounds 5h, 5m, 5t and ningnanmycin with tobacco mosaic virus coat protein (TMV-CP). The MST results were shown in Fig. 4, where the binding of compounds 5h, 5m, 5t and ningnanmycin to TMV-CP protein yielded Kd values of 0.499, 0.008, 69.625 and 2.338 µmol/L, respectively. As displayed in MST results that the combining capacity in the following order of 5m > 5h > ningnanmycin > 5t was basically consistent with the antiviral activity, it shows that there was a good interaction between compound 5m and TMV-CP.
MST results of compounds 5h (A), 5m (B), 5t (C) and ningnanmycin(D).
3.2.3 Molecular docking of compounds 5m and 5t with TMV-CP
The molecular docking was studied that 5m and 5t with TMV-CP (PDB code: 1EI7) were analyzed using Discovery Studio 4.5 Client and the binding results were clearly shown in Fig. 5. Compounds 5m and 5t were combined with TMV-CP in one pocket respectively. Among of them, compound 5m forms four hydrogen bonds with ARGB:261, AGRA:134, SERB:255, and LYSB:268, respectively, after docking with TMV-CP, and compound 5t forms two hydrogen bonds with SERB:255 and ARGB:261 after docking. It can be seen that in combination with TMV-CP, compound 5m has two more stable hydrogen bonds than that of 5t, indicating that the antiviral activity of 5m was better than 5t. The interaction between these molecules and TMV-CP may weaken the interaction between the two subunits of TMV-CP, so as to prevent the self-assembly of TMV and weaken the binding ability with TMV-CP.
Molecular docking results of compounds 5m (A and B), 5t (C and D).
3.3 Antifungal activity assay
3.3.1 In vivo antifungal activity of the target compounds
The effects of the target compounds against fungal were evaluated by the mycelial growth inhibition method at 100 µg/mL. The data in Table 3 showed that some of the target compounds exhibited good antifungal activity. Among them, the inhibitory activities of compounds 5i and 5t against CT were 73.2 and 71.0%, respectively, which were similar to that of azoxystrobin (72.5%). The inhibitory activities of compounds 5k (62.2%) and 5u (60.0%) were similar to those of azoxystrobin (61.4%) against CG. Compound 5u (88.6%) showed better activity of anti-RS than azoxystrobin (78.4%). It is noteworthy that most of the target compounds had good inhibitory effects against PL. The inhibitory rates of compounds 5i and 5p were 81.5 and 87.3%, respectively, close to azoxystrobin (86.3%). Compounds 5k, 5q, 5s and 5t exhibited significant activity against PL, with values of 90.1, 93.8, 96.3 and 96.0% respectively, which were exceeded that of azoxystrobin (86.3%). To further confirm the antifungal activity of the target compound, EC50 tests were performed on some compounds, and the test results were shown in Table 4. The EC50 values of the tested compounds were close to or better than the corresponding control azoxystrobin. It was noteworthy that EC50 values of compound 5k (26.0 µg/mL) against RS were comparable to azoxystrobin (26.7 µg/mL), and compounds 5t and 5s display remarkable activity against PL, with EC50 values of 6.9 and 10.1 µg/mL (Fig. 6), respectively, which were superior compared to azoxystrobin (10.8 µg/mL).
Compounds
Inhibition rate (%)a
AB
FF
FO
CT
PC
CG
RS
FG
PS
PP
BC
PL
5a
25.0 ± 4.3
18.3 ± 1.1
7.8 ± 1.3
12.7 ± 0.3
21.9 ± 2.2
25.8 ± 1.6
38.6 ± 2.8
19.7 ± 1.6
32.8 ± 1.9
11.8 ± 2.3
36.1 ± 0.1
–
5b
17.5 ± 1.9
13.4 ± 2.8
6.5 ± 2.3
25.0 ± 0.2
21.4 ± 2.8
42.5 ± 1.6
65.3 ± 1.7
14.0 ± 3.2
33.7 ± 0.6
25.4 ± 2.3
25.0 ± 2.7
76.3 ± 3.1
5c
18.8 ± 1.7
29.8 ± 2.1
6.5 ± 1.8
19.7 ± 0.5
17.9 ± 3.0
22.3 ± 3.5
41.6 ± 2.3
21.9 ± 1.0
28.5 ± 1.7
8.7 ± 1.8
19.0 ± 0.5
20.1 ± 3.9
5d
5.2 ± 2.8
17.2 ± 2.8
5.2 ± 1.1
5.2 ± 0.1
5.2 ± 2.8
5.2 ± 1.7
5.2 ± 3.1
5.2 ± 0.8
5.2 ± 4.2
5.2 ± 0.1
24.3 ± 4.4
5.2 ± 1.4
5e
28.0 ± 1.9
36.7 ± 2.8
9.2 ± 3.0
17.1 ± 0.4
7.4 ± 1.5
18.8 ± 0.8
17.5 ± 1.9
25.0 ± 1.7
24.1 ± 3.7
16.6 ± 2.2
18.0 ± 3.6
18.8 ± 3.1
5f
22.3 ± 2.4
27.5 ± 3.8
3.5 ± 1.1
21.0 ± 0.1
10.0 ± 1.6
26.3 ± 1.9
41.2 ± 4.6
21.9 ± 2.2
25.4 ± 2.4
12.7 ± 2.6
37.2 ± 1.4
17.5 ± 2.8
5g
20.1 ± 2.8
12.2 ± 3.3
1.7 ± 2.6
23.6 ± 0.3
22.8 ± 2.9
24.1 ± 2.1
45.6 ± 1.9
13.6 ± 1.6
28.5 ± 4.0
16.6 ± 3.6
24.4 ± 2.3
77.3 ± 0.1
5h
32.0 ± 0.7
22.9 ± 1.2
3.9 ± 1.2
17.9 ± 0.2
29.8 ± 3.6
23.6 ± 2.3
63.1 ± 1.4
19.3 ± 2.9
21.0 ± 0.1
23.2 ± 1.6
31.0 ± 0.3
77.3 ± 0.2
5i
24.1 ± 0.9
29.8 ± 2.8
7.4 ± 2.1
73.2 ± 0.2
19.3 ± 4.1
49.1 ± 2.2
64.4 ± 2.8
6.5 ± 1.0
26.7 ± 4.0
35.0 ± 1.1
16.3 ± 3.6
81.5 ± 2.1
5j
43.8 ± 4.3
32.5 ± 2.1
12.2 ± 2.2
63.1 ± 0.1
36.4 ± 3.9
45.6 ± 2.9
67.5 ± 2.1
24.5 ± 0.8
23.2 ± 3.4
15.7 ± 3.7
19.8 ± 1.8
40.7 ± 1.1
5k
30.2 ± 4.3
29.1 ± 2.7
3.5 ± 1.1
48.6 ± 0.3
17.1 ± 1.5
62.2 ± 1.1
68.8 ± 4.1
12.2 ± 2.2
25.1 ± 4.3
31.8 ± 0.2
25.5 ± 0.9
90.1 ± 4.3
5l
19.7 ± 2.4
19.1 ± 4.9
20.6 ± 2.5
28.5 ± 0.3
21.4 ± 2.8
27.1 ± 1.9
39.4 ± 2.1
–
32.4 ± 4.1
22.8 ± 4.1
28.1 ± 3.3
38.1 ± 2.7
5m
17.1 ± 4.3
28.7 ± 4.8
22.8 ± 4.3
25.8 ± 0.3
17.1 ± 2.3
37.7 ± 1.1
56.5 ± 3.8
22.8 ± 1.6
22.3 ± 3.2
45.1 ± 2.6
34.2 ± 0.5
51.7 ± 1.7
5n
19.3 ± 2.4
29.1 ± 1.4
21.4 ± 3.2
26.7 ± 0.2
27.1 ± 0.2
32.8 ± 1.0
49.5 ± 1.7
34.2 ± 0.1
27.1 ± 3.5
13.6 ± 1.6
24.7 ± 1.1
55.2 ± 2.7
5o
18.4 ± 3.3
24.5 ± 2.4
23.2 ± 2.0
24.1 ± 0.2
14.0 ± 1.8
27.6 ± 1.7
48.2 ± 1.9
12.2 ± 1.6
14.9 ± 2.8
28.5 ± 0.9
33.2 ± 2.2
75.8 ± 1.5
5p
12.2 ± 1.9
29.1 ± 3.0
18.8 ± 1.4
11.4 ± 0.3
7.4 ± 1.5
22.3 ± 1.6
41.2 ± 1.2
–
27.1 ± 5.4
21.0 ± 3.1
35.4 ± 4.8
87.3 ± 0.1
5q
20.1 ± 1.2
14.9 ± 4.6
17.1 ± 4.1
34.6 ± 0.2
21.0 ± 2.5
27.1 ± 1.1
48.6 ± 1.2
6.5 ± 1.7
–
16.6 ± 1.1
17.0 ± 0.9
93.8 ± 0.2
5r
22.8 ± 2.4
26.4 ± 2.9
18.4 ± 1.9
30.2 ± 0.7
27.1 ± 3.5
32.0 ± 3.1
41.2 ± 1.2
22.3 ± 1.7
20.6 ± 4.0
25.4 ± 4.6
34.4 ± 4.2
48.6 ± 3.4
5s
25.8 ± 1.7
46.3 ± 1.0
28.9 ± 1.3
17.1 ± 0.1
40.7 ± 1.4
30.2 ± 4.0
67.3 ± 0.2
52.6 ± 5.0
34.2 ± 0.2
64.9 ± 1.1
19.8 ± 2.8
96.3 ± 1.2
5t
34.6 ± 1.7
29.8 ± 3.6
19.7 ± 2.3
71.0 ± 0.2
31.1 ± 3.6
56.5 ± 1.1
71.9 ± 1.2
21.0 ± 0.8
21.0 ± 3.2
45.1 ± 1.6
22.1 ± 1.8
96.0 ± 1.7
5u
65.7 ± 2.0
42.1 ± 1.5
25.0 ± 1.8
55.2 ± 0.4
49.1 ± 4.2
60.0 ± 3.7
88.6 ± 1.2
46.0 ± 2.2
–
53.9 ± 1.2
31.7 ± 0.4
79.8 ± 3.7
5v
36.8 ± 0.1
45.2 ± 2.7
–
36.8 ± 0.1
42.1 ± 4.5
48.6 ± 1.8
58.3 ± 3.4
28.9 ± 1.3
35.0 ± 3.3
22.8 ± 2.3
45.7 ± 3.6
42.1 ± 2.9
Azoxystrobinb
79.5 ± 4.9
55.8 ± 3.1
59.2 ± 1.5
72.5 ± 2.1
62.4 ± 4.1
61.4 ± 1.5
78.4 ± 1.1
73.6 ± 1.8
56.3 ± 4.1
38.9 ± 3.3
62.5 ± 2.2
86.3 ± 4.1
Compounds
EC50 (µg/mL)a
CG
RS
PL
CT
5i
–
–
–
16.1
5k
40.1
–
33.7
–
5q
–
–
28.4
–
5s
–
–
6.9
23.9
5t
–
–
10.1
–
5u
69.1
26.0
–
–
Azoxystrobinb
57.6
26.7
10.8
12.8
Antifungal activities of compounds 5s and 5t against PL in vitro.
The antifungal activity of the target compounds was also affected by different substituents on R1 and R2. For example, compound 5i (R1 = CH3, R2 = 6-Cl-3-Py) and 5t (R1 = H, R2 = 6-Cl-3-Py) showed good inhibitory activity against CT, which were 73.2% and 71.0%, respectively. It is indicating that when R2 was 6-Cl-3-Py group, the growth of CT could be inhibited, and when R1 was CH3 group could increase the anti-CT activity. In the anti-RS activity data, it can be found that CH3, 6-Cl-3-Py, CH2CH = CH, and 2-Cl-5-thiazoly at the R2 group, the inhibitory activity of compounds against RS was mostly better than compounds with R2 of benzyl substitution. And when R1 was H, it helps to improve the anti-RS activity. Most of the target compounds had significant antifungal activities for against PL. For instance, when R1 was H, compounds 5s (R2 = CH3), 5t (R2 = 6-Cl-3-Py), 5q (R2 = 2-Cl-6-F-Ph) and 5p (R2 = 4-NO2-Ph) were 96.3, 96.0, 93.8 and 87.3%, respectively. It is speculated that when the R1 group was H, and the substitution activity of the R2 group was methyl > six-membered heterocycle > benzene. When R1 was a CH3 group, 5i (R2 = 6-Cl-3-Py) and 5k (R2 = 2-Cl-5-thiazoly), the inhibitory activity against PL were 81.5% and 90.1%, respectively. When R2 was a 2-Cl-5-thiazoly group, it could enhance the anti-PL activity of target compounds. Moreover, it was found that compounds 5i and 5t had good inhibitory activities against the above three strains, and it was inferred that the antifungal spectrum of the target compound could be improved when R2 group was replaced by 6-Cl-3-Py.
3.3.2 Inhibitory effect of compound 5u on RS sclerotia germination and formation
Fig. 7A and 7B indicated that the sclerotia germination inhibition of compound 5u on the RS. Compound 5u and azoxystrobin exhibited general inhibitory activity in a dose-dependent manner. In a word, compound 5u showed better inhibitory effect than azoxystrobin with the inhibition of situation 5u (100 µg/mL) > azoxystrobin (100 µg/mL) > 5u (50 µg/mL) > azoxystrobin (50 µg/mL) > azoxystrobin (10 µg/mL) > 5u (10 µg/mL). As shown in Fig. 7C and 7D, for treatment with compound 5u and azoxystrobin at the concentrations of 100 and 50 µg/mL, the inhibition of sclerotia formation rate reached 100%. The inhibition rate of compound 5u on sclerotia formation was 53.6%, but azoxystrobin was still 100%, when the treatment concentrations of 10 µg/mL. The results indicated that compound 5u could inhibited the sclerotia germination of RS. In summary, these results proved that compound 5u could inhibit the sclerotia germination and formation of RS, and reduce the source infection of RS, so as to control the disease.
Inhibitory activities of compound 5u on sclerotia germination (A, B) and formation (C, D) of RS.
3.3.3 The control efficacy against rice sheath blight disease of compounds 5u on rice leaves
In order to further understand the in vivo activity of compound 5u against rice sheath blight disease, the detached leaf assay was adopted. The Table 5 and Fig. 8 showed that the control efficacies were 55.4 and 75.4%, at 100 and 200 µg/mL, respectively, which were lower than those of azoxystrobin (72.3 and 93.8%). These results indicated that compound 5u had a certain control efficacy on rice sheath blight disease.
Treatment
Concentration (µg/mL)
Lesion length (cm)a
Control efficacy (%)
5u
100
2.9 ± 0.3
55.4
200
1.6 ± 0.2
75.4
Azoxystrobin
100
1.8 ± 0.1
72.3
200
0.4 ± 0.4
93.8
Negative control
–
6.5 ± 0.3
–
The protection efficacy of compound 5u against rice sheath blight disease on detached rice leaves. (A) 5u at 100 μg/mL; (B) 5u at 200 μg/mL; (C) Negative control; (D) Azoxystrobin at 100 μg/mL; (E) Azoxystrobin at 200 μg/mL.
3.3.4 Effect of compound 5u on the hyphae morphology of RS
The effects of compound 5u on the morphology of RS were observed by scanning electron microscopy (SEM). As shown in Fig. 9, the mycelium surface of untreated blank group was smooth and full, presenting a complete cylindrical shape, and good mycelium extension. In sharp contrast to the mycelium treated with compound 5u, when the treatment was 50 µg/mL, part of the mycelium broke, and its surface shrank, folded and collapsed. When the concentration of treatment increased to 100 µg/mL, hyphae ruptured was intensified, and all hyphae showed shrinkage, collapse and distortion. It is speculated that compound 5u might inhibited hyphae reproduction by destroying the epidermal tissue and cell membrane of RS.
Scanning electron micrographs of the hyphae of RS with or without compound 5u treatment. (A, B) control groups (DMSO); (C, D) 5u at 50 µg/mL and (E, F) 5u at 100 µg/mL.
4 Conclusion
In summary, we designed and synthesized a series of novel trifluoromethyl pyrimidine derivatives, and determined their structures by NMR, HRMS and single crystal diffraction. Bioassay results showed that some of the target compounds showed good antiviral and antifungal activities. Especially, the antiviral activity of compound 5m was significantly better than that of ningnanmycin. The interaction mechanism between 5m and TMV, and molecular docking were further studied, and the results were consistent with the experimental results in vivo. Then, the mycelial growth rate method was used to evaluate the inhibitory activity of the target compounds against 12 kinds of fungi at the treatment concentration of 100 µg/mL. The anti-RS activity of compound 5u was equivalent to azoxystrobin, and its mechanism of action had been preliminarily studied. The current research had laid the foundation for the application of trifluoromethyl pyrimidine derivatives in pesticides.
Acknowledgement
The authors gratefully acknowledge the National Nature Science Foundation of China (No. 31701821), the Science Foundation of Guizhou Province (No. 20192452), Natural Science Research Project of Guizhou Education Department (No. 2018009), Frontiers Science Center for Asymmetric Synthesis and Medicinal Molecules, Department of Education, Guizhou Province (No. 2020004), Program of Introducing Talents of Discipline to Universities of China (111 Program, D20023).
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Appendix A
Supplementary material
Supplementary data to this article can be found online at https://doi.org/10.1016/j.arabjc.2022.104110.
Appendix A
Supplementary material
The following are the Supplementary data to this article:Supplementary Data 1
Supplementary Data 1
