Investigation of Novel Pesticides with Insecticidal and Antifungal Activities: Design, Synthesis and SAR Studies of Benzoylpyrimidinylurea Derivatives
by
Peiqi Chen
1,2,3, 
Xiangmin Song
1,2,
Yongmei Fan
1,2,
Weihao Kong
1,2,
Hao Zhang
3 and
Ranfeng Sun
1,2,* 1
Key Laboratory of Green Prevention and Control of Tropical Plant Diseases and Pests, Hainan University, Haikou 570228, China
2
Institute of Tropical Agriculture and Forestry, Hainan University, Haikou 570228, China
3
School of Chemical & Chemical Engineering, Inner Mongolia University, Hohhot 010021, China
*
Author to whom correspondence should be addressed.
Molecules 2018, 23(9), 2203; https://doi.org/10.3390/molecules23092203
Submission received: 9 August 2018
/
Revised: 25 August 2018
/
Accepted: 28 August 2018
/
Published: 31 August 2018
(This article belongs to the Section Medicinal Chemistry)
Abstract
:In order to find pesticides with insecticidal and antifungal activities, a series of novel benzoyl pyrimidinylurea derivatives were designed and synthesized. All target compounds were identified by 1H-NMR spectroscopy and HRMS. Insecticidal and antifungal activity of these compounds were evaluated and the structure-activity relationships (SAR) were clearly and comprehensively illustrated. Compound 7, with low toxicity to zebrafish (LC50 = 378.387 µg mL−1) showed 100% inhibition against mosquito (Culex pipiens pallens) at 0.25 µg mL−1. Both compounds 19 and 25 exhibited broad-spectrum fungicidal activity (>50% inhibitory activities against 13 phytopathogenic fungi), which were better than those of the commercial pesticide pyrimethanil (>50% inhibitory activities against eight phytopathogenic fungi). Furthermore, compounds 19 and 25 exhibited protective activity against Sclerotinia sclerotiorum on leaves of Brassica oleracea L. during in vivo experiments.
1. Introduction
Plant disease and pest control are at least as old as agriculture because there is a need to keep crops free of pests and phythopatogenic fungi. Benzoylphenylureas (BPUs) have been developed as chitin synthesis inhibitors because of their unique mode of action coupled with a high degree of activity on targeted pests and low toxicity to non-target organisms [1,2]. The first commercial product Dimilin (diflubenzuron) (Figure 1), introduced in the market at 1975, exhibits broad spectrum larvicidal activities against caterpillars (Pieris brassicae), mosquitoes (Aedes aegypti), houseflies (Musca domestica), desert locust (Schistocera gregaria) and cotton strainers (Dysdercus superstitiosus) [3]. In the past few years, we and our co-workers have designed and synthesized several series of benzoylphenylureas (A and B, Figure 1) and most compounds exhibited excellent insecticidal activities against oriental armyworm (Mythimna separata) or mosquito (Culex pipiens pallens) [4,5,6,7,8]. Interestingly, compounds C and D (Figure 1) with a 2,2,2-trifluoroethyl group (R = CH2CF3) substituent, displayed much better insecticidal activities against oriental armyworm than other derivatives.
At the same time, a class of phenylurea compounds (E, Figure 2) containing a pyrimidine ring attracted our attention. When bearing two 2,2,2-trifluoroethyl groups on the pyrimidine ring most of these compounds have good insecticidal activity performance against second-instar corn earworm (Helicoverpa zea) and beet armyworm (Spodoptera exigua) at 50 µg mL−1.
More importantly, the 2-aminopyrimidinyl unit is commonly found in the chemical structure of antifungal compounds. For example, the antifungal activity of commercial pyrimethanil (Figure 2) against Alternaria kikuchiana Tanaka was 100% at 0.1 µg mL−1 [9,10,11,12]. Compound F (EC50 = 1.03 µg mL−1) exhibited better antifungal activity against Phytophthora capsici than dimethomorph (EC50 = 4.26 µg mL−1) (Figure 2) [13]. Compound G (Figure 2) with excellent antifungal activity (EC50 = 0.84 µg mL−1) against Phytophthora capsici was discovered in 2011 by Nam et al. [14].
Based on our previous works [4,5,6,7,8] and the literature [9,10,11,12,13,14], in order to find new pesticides with powerful insecticidal and antifungal properties, we were interested in introducing pyrimidine group into the benzoylurea backbone to design a series of benzoylpyrimidinylurea derivatives (Figure 3). Therefor target compounds 1–38 were synthesized and their insecticidal and antifungal activities were evaluated. At the same time, the aquatic toxicity of compounds 7 and 25 were evaluated by a zebrafish embryo acute toxicity test (OECD TG 236).
2. Results
2.1. Synthesis
In order to comprehensively analyze the structure-activity relationship of compounds, the chemical structures of the benzoylpyrimidinylureas was separated into three units, namely, the benzoyl ring (A), pyrimidine ring (B) and urea bridge (C) (Figure 3). Substituted 2-aminopyrimidines Ia–p were synthesized and used as starting materials for the synthesis of the target compounds (Scheme 1 and Scheme 2).
In a first series of compounds, intermediate Ia was retained and the benzoyl moiety was altered resulting in compounds 1 to 10 (Scheme 3).
Secondly, different substituents were changed on the pyrimidine ring and the compounds 11–30 with substituted 2-aminopyrimidines were obtained (Scheme 4).
Thirdly, we replaced the oxygen atom on the urea bridge with sulfur or nitrogen to gain compounds 31 and 32 (Scheme 5).
Finally, the benzoyl ring was exchanged by a six- or five-membered nitrogen heterocycles to compare the effects between benzene ring and heterocycles on the bioactivity of the compounds, therefor compounds 33–37 were synthesized (Scheme 6). To illustrate the role of the carbonyl group of the benzoyl group in the biological activity of the target compounds, compound 38 was synthesized from 2,6-dichloroaniline (Scheme 6). All reactions gave excellent yields and the NMR data of compounds proved the proposed structures. Among them, compounds with trifluoromethyl groups have a characteristic quartet peak with 13C-19F couplings in the NMR spectra. The spectra are shown in Figures S1–S101 in the Supporting Information.
2.2. Bioassays
2.2.1. Toxicity against Mosquito (Culex pipiens pallens)
Table 1 shows the larvicidal activities of the compounds 1–25, 31, 37 and fipronil against mosquito. The bioassay results indicated that eighteen compounds (1–7, 9–10, 13–14, 16, 18, 20, 22–24, 31) exhibited more than 50% larvicidal activities against mosquito and eleven of them reached 100% at 10 µg mL−1, especially, compound 7 which showed 100% mortality even at 0.25 µg mL−1. By comparing the chemical structure of compounds 2 (2-Cl), 3 (2-Br) and 4 (2-I), we found that the larvicidal activitives decreased (100%, 60% mortality at 2 µg mL−1, 50% mortality at 10 µg mL−1 respectively) as the radius of the halogen atom at the 2-position on the benzoyl ring increased. Next, we found that when the 2,6-position on the benzoyl ring was substituted by two halogens or electron-rich groups, it was not conducive to larvicidal activity, for example, the larva mortalities of compounds 8 (2-OCH3), 9 (2,4-di-Cl) and 10 (2,6-di-Cl) were 20% at 10 µg mL−1, 40% at 2 µg mL−1 and 20% at 5 µg mL−1 respectively.
However, electron-withdrawing groups in the para position enhanced the bioactivity of the compounds, for example, the larva mortality of compound 7 (4-CF3) reached 100% at 0.25 µg mL−1. When the 4,6-position on the pyrimidine ring were substituted with haloalkoxyl, alkoxy or alkyl groups, the larvicidal activity decreased successively, at the same time the longer the alkoxy chain, the larvicidal activity was better. For example, the mortalities of compounds 11 (4,6-di-CH3), 12 (4,6-di-OCH3), 13 (4,6-di-OEt) and 2 (4,6-di-OCH2CF3) were 10% at 10 µg mL−1, 20% at 10 µg mL−1, 70% at 2 µg mL−1, 100% at 0.5 µg mL−1, respectively. Similarly, the structure-activity relationships of compounds 2 and 14 told us that a trifluoroethoxy group was more helpful to increase the mortality activities than s halogen group. Furthormore it was easy to find that the urea bridge unit was important to the larvicidal activity. For example compound 31 containing a thiourea unit (50% mortality at 1 µg mL−1) exhibited lower larvicidal activity than compound 2 (100% mortality at 0.5 µg mL−1). Finally, when a pyridine ring (compound 37) was employed instead of a benzene ring (compound 2), the larvicidal activities were reduced greatly (15% mortality of compound 37 at 10 µg mL−1 and 100% mortality of compound 2 at 0.5 µg mL−1). Therefore, it was concluded that electron-withdrawing groups (such as a trifuloromethyl group) on the para position of the benzoyl ring, alkoxy groups (especially haloalkoxyl groups, such as a trifluoroethoxy group) and a urea bridge unit favored the larvicidal activities.
2.2.2. Stomach Toxicity against Oriental Armyworm (Mythimna separata)
The bioactivity results indicated that most compounds exhibited certain insecticidal activities against oriental armyworm as listed in Table 1. Firstly, we can easily see from compounds 2 (2-Cl) and 5 (4-Cl) or 6 (2-CF3) and 7 (4-CF3) and their larva moralities (5%, 65%, 15% and 65% at 600 µg mL−1 respectively) that para-substituents of the benzoyl ring result in better insecticidal activities than ortho-substituents. Secondly, it was figured out that both the radius and electronegativity of the halogen atom at the 2-position on the benzoyl ring influenced the larvicidal activities, for example, compound 3 (2-Br, 100% mortality at 200 µg mL−1) was a more effective insecticidal agent than compound 4 (2-I, 70% mortality at 600 µg mL−1) which exhibited much better larvicidal activity than compound 2 (2-Cl, 5% mortality at 600 µg mL−1). At the same time, it also pointed out that chlorine atom on the pyrimidine ring was beneficial to improve the insecticidal activity of compounds, for example, the larva mortalities of compounds 14 (4,6-Cl), 15 (4-Cl, 6-CH3) and 16 (4-Cl, 6-OCH3) were 50%, 30%, 5% at 600 µg mL−1 respectively. Interestingly, when we replaced the oxygen atom on the urea bridge with a sulfur atom, the insecticidal activities of the compounds increased greatly, as seen by comparing the insecticidal activities of compound 2 (5% mortality of at 600 µg mL−1) and compound 31 (100% mortality at 600 µg mL−1).
2.2.3. In Vitro Antifungal Activity
The fungicidal results are listed in Table 2. Most of the derivatives showed antifungal activity against 14 kinds of phytopathogenic fungi. We first determined the antifungal activities of compounds 2 (2-Cl), 3 (2-Br), 4 (2-I), 5 (4-Cl), 6 (2-CF3), 7 (4-CF3) and 8 (2-OCH3, 5-Cl) and we found that ortho-halogen substitutents on the benzoyl ring were beneficial for antifungal activity. Among them, compound 2 was better than the others. At this moment, our design strategy was divided into two parts. One was to maintain 2-chloro group on the benzoyl ring and a series of differently substituted compounds (such as compounds 11–22) at the 4,6-position on the pyrimidine ring were synthesized. The other was that we continued to explore the effect of the number of halogen atoms on the benzoyl ring for the antifungal activity, followed by the synthesis of compounds with different substitutions at the 4,6-position on pyrimidine ring, such as compounds 1, 9, 10 and 23–30.
From the first part of strategy, we reached three important conclusions. Firstly, compounds with alkoxy groups had better antifungal activity than those bearing an alkyl group, and if the hydrogen atom on the alkoxy group was substituted with a halogen atom, the antifungal activity of compounds would be further enhanced. For example, the antifungal activity order of compounds 2, 11, 12 and 13 was compound 2 (4,6-di-OCH2CF3) > compound 13 (4,6-di-OEt) > compound 12 (4,6-di-OCH3) > compound 11 (4,6-di-CH3) (Table 2). Secondly, chlorine atoms were beneficial to increase the antifungal activity of the compounds. For example, the antifungal activity order of compounds 14, 15 and 16 was compound 14 (4,6-Cl) > compound 15 (4-Cl, 6-CH3) > compound 16 (4-Cl, 6-OCH3) (Table 2).
Thirdly, when the 4,6-positions of the pyrimidine ring were substituted by a chlorine atom and trifluoroethoxy group (such as compound 17), the antifungal activity was weakened instead of enhanced. Therefore, we further fixed the 4-position of the pyrimidine ring with a chlorine atom or trifluoroethoxy group, and a series of compounds with nitrogen-substituted group at 6-position of the pyrimidine ring were synthesized, such as compounds 18 (4-Cl, 6-morpholine), 19 (4-Cl, 6-N’,N’-diethyl), 21 (4-OCH2CF3, 6-pyrrolidinyl) and 22 (4-OCH2CF3, 6-N’-hexyl) (Table 2). Fortunately, compound 19 exhibited broad-spectrum fungicidal activity (>60% inhibitory activities against 13 phytopathogenic fungi), which was better than the inhibitory activities of commercial pyrimethanil (>50% against eight phytopathogenic fungi, Table 2).
As another part of our strategy, we found that the antifungal activity of 10 (2,6-Cl on the benzoyl ring) is superior to compounds 9 (2,4-Cl on the benzoyl ring, Table 2). Compound 25 exhibited broad-spectrum fungicidal activity (>50% inhibitory activities against 13 phytopathogenic fungi). Especially notable was the inhibition activity of compound 25 against Rhizoctonia cerealis, Phytophthora capsica, Botrytis cinereal and Physalospora piricola. reached 94.9%, 90.7%, 89.7% and 84.2% at 50 µg mL−1 respectively, which was comparable to commercial pyrimethanil (Table 2). Unlike the low activity of compound 17 (2-Cl), the broad-spectrum antifungal activity of compound 25 indicated that 2,6-dichloro substitution on the benzoyl ring was very important for the antifungal activity. However, when we replaced the ethyl group on the nitrogen with benzyl group (compound 29 compared with compound 27) or the 5-position of the pyrimidine ring was substituted with 2-butyl group (compound 30), the antifungal activity decreased significantly (Table 2). This result indicated that groups with large steric hindrance were not conducive to antifungal activity.
In the investigation of the urea bridge unit, a benzoylthiourea derivative (compound 31) and a benzoylguanidine derivative (compound 32) displayed lower antifungal activities than compound 2 and compound 27, respectively (Table 2). Although the phenylurea compound 38 also showed better antifungal activity (>50% inhibitory activities against 10 phytopathogenic fungi), its antifungal activity was lower and its antifungal spectrum smaller than those of compound 19. Finally, because of the broad spectrum and powerful antifungal activity of compounds 19 and 25, the values of EC50 for compounds 19 and 25 against Sclerotinia sclerotiorum, Botrytis cinereal, Physalospora piricola, Rhizoctonia cerealis were determined and are listed in Table 3.
2.2.4. In Vivo Antifungal Bioassay against Sclerotinia sclerotiorum
The protective effets of compounds 19 and 25 and carbendazim (positive control) against S. sclerotiorum on detached Brassica oleracea L. leaves were evaluated. The results showed that both compounds 19 and 25 exhibited protective activity on leaves of Brassica oleracea L. (Table 4).
This protective effect was enhanced with the increasing concentration of the compounds (experimental photos shown in Figure S102 in the Supporting Information). For example, the values of the protective effect for compound 19 were 55.7% at 3 mg mL−1, 35.2% at 1.5 mg mL−1, 34.1% at 0.5 mg mL−1 and the protective effect values for compound 25 were 83.0% at 3 mg mL−1, 35.2% at 1.5 mg mL−1, 20.5% at 0.5 mg mL−1 (Table 4). Interestingly, although compound 19 has better in vitro antifungal activity than compound 25, it appears that compound 25 has a better protective effect.
2.2.5. Zebrafish Embryo Toxicity Assay
The fish embryo acute toxicity test (FET) with zebrafish embryos, which is a general model in ecotoxicology and toxicology had been established in our labs [15,16]. The mortality in the positive control was 40% greater than 30% and survival in the negative control was 100%. As shown in Table 5, compound 7 has low toxicity to zebrafish embryos (LC50 = 378.387 μg mL−1), which was what we like to see because of its excellent larvicidal activity to mosquito. At the same time, compound 25 which exhibited broad-spectrum antifungal activity also possesses low toxicity to zebrafish embryos (LC50 = 21.668 μg mL−1). Fortunately, both compounds 7 and 25 did not affect zebrafish embryo hatching, induce the pericardial cysts and produce abnormality (such as malformation and coagulation) during the zebrafish embryo acute toxicity test.
3. Materials and Methods
3.1. Instruments
1H-NMR and 13C-NMR spectra were obtained at 500/126 MHz using a Bruker Avance III 500 spectrometer (Bruker Daltonics, Bremen, Germany) in CDCl3 or DMSO-d6 solution with tetramethylsilane as the internal standard. Chemical shift values (δ) are given in ppm. HRMS data were obtained on FTICR-MS (Ionspec 7.0 T, Lebrilla League, Davis, CA, USA) high resolution mass spectrometer and LQC Advantage MAX multi-stage ion mass spectrometer (Agilent Technologies Inc., CA, USA). The melting points were determined on an X-4 binocular microscope melting point apparatus (Beijing Tech Instruments Co., Beijing, China) and are uncorrected. Yields were not optimized.
3.2. General Synthesis
All anhydrous solvents were dried and purified by standard techniques. Chemicals (analytical grade) were purchased from Aladdin (Shanghai, China).
3.3. General Synthesis Procedure for Substituted 2-Aminopyrimidines Ia–c
Intermediates Ia–c were prepared according to the literature [17]. A solution of NaH (0.35 g, 14.6 mmol) in dry THF (12 mL) was cooled to 0 °C under N2, then a substituted alcohol (15.2 mmol) was added dropwise. The mixture was stirred for 15 min while maintaining the temperature at 0 °C. Next, 2-amino-4,6-dichloropyrimidine (1.0 g, 6.1 mmol) was added to the solution. The reaction was continued at 62 °C for 15 h. Then the mixture was cooled to ambient temperature, and quenched with 1 mL of 1 M hydrochloric acid solution. The mixture was diluted with EtOAc (20 mL), washed twice with a saturated NaHCO3 solution (20 mL) and brine (20 mL), dried with anhydrous Na2SO4 and evaporated in vacuo. Finally, the residue was purified by silica gel column chromatography (EtOAc/petroleum ether) to afford compounds Ia–c.
4,6-Bis(2,2,2-trifluoroethoxy)pyrimidin-2-amine (Ia). Yellow oil, yield = 94%. 1H-NMR (CDCl3) δ 5.58 (s, 1H, ArH), 4.96 (s, 2H, NH2), 4.57 (q, J = 8.6 Hz, 4H, OCH2CF3).
4,6-Dimethoxypyrimidin-2-amine (Ib). White solid, mp 87–89 °C, yield = 91%. 1H-NMR (CDCl3) δ 5.46 (s, 1H, ArH), 5.09 (s, 2H, NH2), 3.84 (s, 6H, OCH3).
4,6-Diethoxypyrimidin-2-amine (Ic). White solid, mp 174–176 °C, yield = 94%. 1H-NMR (CDCl3) δ 5.42 (s, 1H, ArH), 4.95 (s, 2H, NH2), 4.23 (q, J = 7.1 Hz, 4H, OCH2CH3), 1.35 (t, J = 7.1 Hz, 6H, OCH2CH3).
3.4. General Synthesis Procedure for Substituted 2-Aminopyrimidines Id–f
Intermediates Id–f were prepared by a method similar to that for intermediates Ia–c. A solution of NaH (5.8 mmol) in dry THF (12 mL) was cooled to 0 °C under N2, then a substituted alcohol (6.1 mmol) was added dropwise. The mixture was stirred for 15 min while maintaining the temperature at 0 °C. Next, 2-amino-4-chloro-6- substituted-pyrimidine (5.8 mmol) was added to the solution. The reaction was continued at 62 °C for 15 h. Then the mixture was cooled to ambient temperature, and quenched with 1 mL of 1 M hydrochloric acid solution. The mixture was diluted with EtOAc (20 mL), washed twice with a saturated NaHCO3 solution (20 mL) and brine (20 mL), dried with anhydrous Na2SO4 and evaporated in vacuo. Finally, the residue was purified by silica gel column chromatography (EtOAc/petroleum ether) to afford compounds Id–f.
4-Chloro-6-methoxypyrimidin-2-amine (Id). White solid, mp 165–167 °C, yield = 84%. 1H-NMR (CDCl3) δ 6.04 (s, 1H, ArH), 5.35–5.14 (m, 2H, NH2), 3.81 (s, 3H, OCH3).
4-Methyl-6-(2,2,2-trifluoroethoxy)pyrimidin-2-amine (Ie). White solid, mp 109–111 °C, yield = 69%. 1H-NMR (CDCl3) δ 6.42 (s, 1H, ArH), 5.50 (s, 2H, NH2), 4.60 (q, J = 8.4 Hz, 2H, OCH2CF3), 2.18 (s, 3H, CH3).
4-Chloro-6-(2,2,2-trifluoroethoxy)pyrimidin-2-amine (If). White solid, mp 82–83 °C, yield = 76%. 1H-NMR (CDCl3) δ 6.23 (s, 1H, ArH), 5.59 (s, 2H, NH2), 4.70 (q, J = 8.4 Hz, 2H, OCH2CF3).
3.5. General Synthesis Procedure for Substituted 2-Aminopyrimidines Ig–m
Intermediates Ig–m were prepared according to [18]. A suspension of CuI (5.8 mg, 0.03 mmol), K2CO3 (0.41 g, 2.9 mmol), the corresponding amine (0.21 g, 2.4 mmol) and substituted 2-aminopyrimidines (0.4 g, 2.4 mmol) in 2 mL DMF was stirred for 30 min at room temperature and then the mixture was heated to 110 °C. At the end of the reaction, as monitored by TLC, the reaction mixture was cooled to room temperature and diluted with 20 mL of EtOAc, washed twice with a saturated NaHCO3 solution (20 mL) and brine (20 mL), dried with anhydrous MgSO4 and evaporated in vacuo. Finally, the residue was purified by silica gel column chromatography to afford compound Ig–m.
4-Chloro-6-morpholinopyrimidin-2-amine (Ig). White solid, mp 210–212 °C, yield = 79%. 1H-NMR (CDCl3) δ 5.94 (s, 1H, ArH), 5.00 (s, 2H, NH2), 3.79–3.68 (m, 4H, morpholine), 3.55 (t, J = 4.9 Hz, 4H, morpholine).
6-Chloro-N4,N4-diethylpyrimidine-2,4-diamine (Ih). White solid, mp 104–106 °C, yield = 81%. 1H-NMR (CDCl3) δ 5.84 (s, 1H, ArH), 5.04 (s, 2H, NH2), 3.42 (s, 4H, CH2CH3), 1.15 (t, J = 7.1 Hz, 6H, CH2CH3).
N4-(Pyrrolidin-1-yl)-6-(2,2,2-trifluoroethoxy)pyrimidine-2,4-diamine (Ii). White solid, mp 118–120 °C, yield = 51%. 1H-NMR (CDCl3) δ 5.21 (s, 1H, ArH), 4.82 (s, 2H, NH2), 4.67 (q, J = 8.7 Hz, 2H, OCH2CF3), 3.38 (s, 4H, tetrahydropyrrole), 1.93 (d, J = 5.9 Hz, 4H, tetrahydropyrrole). 13C-NMR (CDCl3) δ 168.7, 163.3, 162.0, 124.9, 122.7, 62.0, 61.7, 61.4, 61.1, 46.5, 25.3. HRMS (ESI): m/z calcd for C10H13F3N4O [M + H]+: 263.1120, found 263.1136.
N4-Hexyl-6-(2,2,2-trifluoroethoxy)pyrimidine-2,4-diamine (Ij). Yellow oil, yield = 53%. 1H-NMR (CDCl3) δ 5.24 (s, 1H, ArH), 5.13 (s, 1H, NH), 5.06 (s, 2H, NH2), 4.65 (q, J = 8.6 Hz, 2H, OCH2CF3), 3.12 (q, J = 6.7 Hz, 2H, N–CH2(CH2)4CH3), 1.77–1.42 (m, 2H, N–(CH2)4CH2CH3), 1.44–1.11 (m, 6H, N–CH2(CH2)3CH2CH3), 0.88 (t, J = 6.8 Hz, 3H, N–(CH2)5CH3). 13C-NMR (CDCl3) δ 169.4, 165.6, 162.1, 124.7, 122.5, 62.0, 61.7, 61.5, 61.2, 41.8, 31.5, 29.2, 26.6, 22.6, 14.0. HRMS (ESI): m/z calcd for C10H19F3N4O [M + H]+: 293.1589, found 293.1593.
N4,N4-Diethyl-6-(2,2,2-trifluoroethoxy)pyrimidine-2,4-diamine (Ik). Yellow oil, yield = 49%. 1H-NMR (CDCl3) δ 5.32 (s, 1H, ArH), 4.78 (s, 2H, NH2), 4.72–4.61 (m, 2H, OCH2CF3), 3.41 (q, J = 7.0 Hz, 4H, CH2CH3), 1.13 (t, J = 7.1 Hz, 6H, CH2CH3). 13C-NMR (CDCl3) δ 170.4, 169.3, 163.9, 124.9, 81.7, 62.3, 62.0, 61.7, 61.4, 42.1, 12.9. HRMS (ESI): m/z calcd for C10H15F3N4O [M + H]+: 265.1276, found 265.1280.
N4-Benzyl-6-(2,2,2-trifluoroethoxy)pyrimidine-2,4-diamine (Il). Yellow oil, yield = 55%. 1H-NMR (CDCl3) δ 7.36–7.15 (m, 5H, Ph), 5.66 (s, 1H, ArH), 5.22 (s, 1H, NH), 5.02 (s, 2H, NH2), 4.59 (q, J = 8.6 Hz, 2H, OCH2CF3), 4.31 (d, J = 6.0 Hz, 2H, PhCH2). 13C-NMR (CDCl3) δ 169.4, 165.6, 162.2, 138.2, 128.7, 127.5, 127.1, 126.9, 124.7, 122.5, 120.3, 62.0, 61.7, 61.4, 61.2, 45.6. HRMS (ESI): m/z calcd for C13H13F3N4O [M + H]+: 299.1120, found 299.1119.
N4-Benzyl-N4-ethyl-6-(2,2,2-trifluoroethoxy)pyrimidine-2,4-diamine (Im). Yellow oil, yield = 43%. 1H-NMR (DMSO-d6) δ 7.65–7.03 (m, 5H, Ph), 6.23 (s, 2H, NH2), 5.31 (s, 1H, ArH), 4.86 (q, J = 9.2 Hz, 2H, OCH2CF3), 4.69 (s, 2H, PhCH2), 3.41 (s, 2H, CH2CH3), 1.04 (t, J = 7.0 Hz, 3H, CH2CH3). 13C-NMR (DMSO-d6) δ 169.6, 165.1, 163.1, 129.4, 127.9, 127.7, 126.1, 123.9, 75.6, 61.3, 61.1, 42.7, 13.4. HRMS (ESI): m/z calcd for C15H17F3N4O [M + H]+: 327.1433, found 327.1429.
3.6. Synthesis of 5-(sec-Butyl)-4,6-dichloropyrimidin-2-amine (Intermediate In)
Intermediate In was prepared according to [19]. Elemental sodium (0.30 g, 13.0 mmol) was added into absolute ethanol (7 mL) under N2 while being intensively stirred with a magnetic stirrer. After all the sodium was dissolved and the reaction mixture was cooled to room temperature, guanidine hydrochloride (0.49 g, 5.1 mmol) was added under intensive stirring, followed by the diethyl sec-butylmalonate (1.9 g, 4.6 mmol). The reaction mixture was further stirred intensively at room temperature. After another 4 h, absolute ethanol (5 mL) was added and the reaction mixture was refluxed for 1 h. Afterward, ethanol was evaporated on a vacuum rotary evaporator and water (12 mL) was added to the reaction mixture. After stirring, the product was dissolved. The obtained mixture was subsequently neutralized by acetic acid and then this mixture was heated under reflux for 10 min and then cooled to room temperature. This heating and cooling was repeated twice to get a well-filterable solid. The solid was filtered off, washed with water (2 × 50 mL), ethanol (2 × 50 mL), and acetone (2 × 50 mL). The product was dried under high vacuum for 2 days. Subsequently, the aboveobtained solid (4.6 mmol) was suspended under N2 in a solution of (chloro-methylene)dimethyl ammonium chloride (4.8 g, 37.1 mmol) in chloroform (19 mL). The reaction mixture was subsequently heated at reflux for 4 h, during which the starting material was completely dissolved. The reaction mixture was cooled to the room temperature, poured into ice and rapidly neutralized with a saturated aqueous NaHCO3 solution. The obtained mixture was quickly transferred into a separatory funnel and immediately extracted with chloroform (3 × 20 mL). The organic layers were combined together, dried over MgSO4, filtered and concentrated down on a rotary evaporator. This crude residue was dissolved in the mixture of ethanol (9 mL) and 37% aqueous HCl (0.9 mL). The reaction mixture was heated at 50 °C for 2 h. After that, water (14 mL) was added and the reaction mixture was stirred for 10 min. The precipitated product was filtered off and washed with a water/ethanol mixture (v/v = 1/1, 2 × 5 mL), 5 % aqueous solution of NaHCO3 (5 mL). The product was subsequently recrystallized from aqueous ethanol, filtered off, washed with a water/ethanol mixture (v/v = 1/1, 5 mL) and the solid recrystallized from ethanol to give intermediate In as a white solid, mp 157–159 °C, yield = 73%. 1H-NMR (CDCl3) δ 5.98 (d, J = 6.5 Hz, 2H, NH2), 3.27 (dq, J = 6.9, 3.7, 2.9 Hz, 1H, CH), 1.95–1.75 (m, 1H, CHCH2), 1.63 (dddd, J = 16.0, 9.0, 4.7, 2.3 Hz, 1H, CHCH2), 1.24 (dt, J = 7.4, 2.3 Hz, 3H, CHCH3), 0.77 (ddd, J = 9.1, 5.7, 2.1 Hz, 3H, CHCH2CH3).
3.7. Synthesis of 5-(sec-Butyl)-6-chloro-N4,N4-diethylpyrimidine-2,4-diamine (Intermediate Io)
Intermediate Io was prepared by a method similar to that used for compounds Ig–m. Yellow oil, yield = 42%. 1H-NMR (DMSO-d6) δ 6.44 (s, 2H), 3.23 (dd, J = 13.7, 6.9 Hz, 2H), 3.05 (dd, J = 13.7, 7.0 Hz, 2H), 2.79 (q, J = 7.4 Hz, 1H), 1.79–1.61 (m, 2H), 1.31 (d, J = 7.1 Hz, 3H), 1.05 (t, J = 7.0 Hz, 6H), 0.71 (t, J = 7.4 Hz, 3H). 13C-NMR (DMSO-d6) δ 170.8, 160.8, 159.7, 114.1, 45.8, 34.4, 28.3, 19.0, 14.0, 13.6. HRMS (ESI): m/z calcd for C12H21ClN4 [M + Na]+: 279.1352, found 279.1356.
3.8. Synthesis of 5-(sec-Butyl)-4-chloro-6-(2,2,2-trifluoroethoxy)pyrimidin-2-amine (Intermediate Ip)
Intermediate Ip was prepared by a method similar to that used for compounds Id–f. White solid, mp 53–54 °C, yield = 40%. 1H-NMR (DMSO-d6) δ 7.00 (s, 2H, NH2), 4.98 (q, J = 9.0 Hz, 2H, OCH2CF3), 3.02 (dt, J = 9.3, 6.7 Hz, 1H, CH), 1.76–1.62 (m, 1H, CHCH2), 1.56 (ddd, J = 13.6, 7.6, 6.4 Hz, 1H, CHCH2), 1.18 (d, J = 7.1 Hz, 3H, CHCH3), 0.75 (t, J = 7.4 Hz, 3H, CHCH2CH3). 13C-NMR (DMSO-d6) δ 167.6, 160.9, 160.7, 109.8, 62.8, 62.5, 62.2, 62.0, 36.5, 27.9, 19.4, 13.3. HRMS (ESI): m/z calcd for C10H13ClF3N3O [M + H]+: 284.0777, found 284.0799.
3.9. General Synthesis Procedure for Target Compounds 1–30
Compounds 1–30 were synthesized according to [20,21]. Substituted benzamide (5.0 mmol) and 1,2-dichloroethane (10 mL) were added to a 100 mL three-necked flask under N2. The reaction mixture was cooled to 0 °C and oxalyl chloride (1.27 g, 10.0 mmol) was added dropwise under stirring, then the mixture was stirred at room temperature for 1 h, left standing at 60 ° C for 3 h and refluxed for 1 h. The solvent and excess oxalyl chloride were evaporated under reduced pressure to give a yellow transparent liquid. Anhydrous dichloromethane (5 mL) was added to the residue, then a substituted 2-aminopyrimidine (2.5 mmol) was added to the system and reacted at room temperature for 12 h. The solvent was removed under reduced pressure and the residue was purified by flash column chromatography on silica gel to give compounds 1–30.
N-((4,6-bis(2,2,2-Trifluoroethoxy)pyrimidin-2-yl)carbamoyl)-2,6-difluorobenzamide (1). White solid, mp 163–164 °C, yield = 83%. 1H-NMR (DMSO-d6) δ 11.65 (s, 1H, NH), 10.77 (s, 1H, NH), 7.76–7.57 (m, 1H, Ph), 7.26 (t, J = 8.3 Hz, 2H, Ph), 6.40 (s, 1H, ArH), 5.07 (q, J = 8.9 Hz, 4H, OCH2CF3). 13C-NMR (DMSO-d6) δ 170.9, 160.6, 156.3, 148.9, 134.2, 125.6, 123.4, 113.2, 113.0, 86.8, 63.7, 63.4, 63.1, 62.8. HRMS (ESI): m/z calcd for C16H10F8N4O4 [M + H]+: 475.0652, found 475.0641.
N-((4,6-bis(2,2,2-Trifluoroethoxy)pyrimidin-2-yl)carbamoyl)-2-chlorobenzamide (2). White solid, mp 173–175 °C, yield = 95%. 1H-NMR (DMSO-d6) δ 11.47 (s, 1H, NH), 10.96 (s, 1H, NH), 7.65 (dd, J = 7.6, 1.6 Hz, 1H, Ph), 7.61–7.52 (m, 2H, Ph), 7.48 (td, J = 7.3, 1.7 Hz, 1H, Ph), 6.39 (s, 1H, ArH), 5.07 (q, J = 8.9 Hz, 4H, OCH2CF3). 13C-NMR (DMSO-d6) δ 170.9, 169.2, 156.3, 149.0, 135.6, 133.1, 130.7, 130.0, 128.2, 125.7, 123.4, 86.8, 63.6, 63.3, 63.0, 62.8. HRMS (ESI): m/z calcd for C16H11ClF6N4O4 [M + H]+: 473.0451, found 473.0440.
N-((4,6-bis(2,2,2-Trifluoroethoxy)pyrimidin-2-yl)carbamoyl)-2-bromobenzamide (3). White solid, mp 110–112 °C, yield = 77%. 1H-NMR (DMSO-d6) δ 11.46 (s, 1H, NH), 10.96 (s, 1H, NH), 7.73 (d, J = 7.9 Hz, 1H, Ph), 7.62 (dd, J = 7.4, 1.7 Hz, 1H, Ph), 7.57–7.42 (m, 2H, Ph), 6.39 (s, 1H, ArH), 5.07 (q, J = 8.9 Hz, 4H, OCH2CF3). 13C-NMR (DMSO-d6) δ 170.8, 170.0, 156.2, 149.0, 137.8, 133.7, 133.0, 129.8, 128.6, 125.6, 119.5, 86.8, 63.6, 63.3, 63.0, 62.7. HRMS (ESI): m/z calcd for C16H11BrF6N4O4 [M + Na]+: 538.9766, found 538.9769.
N-((4,6-bis(2,2,2-Trifluoroethoxy)pyrimidin-2-yl)carbamoyl)-2-iodobenzamide (4). White solid, mp 115–116 °C, yield = 88%. 1H-NMR (DMSO-d6) δ 11.42 (s, 1H, NH), 11.01 (s, 1H, NH), 7.95 (dd, J = 7.9, 1.0 Hz, 1H, Ph), 7.72–7.38 (m, 2H, Ph), 7.28 (td, J = 7.6, 1.9 Hz, 1H, Ph), 6.39 (s, 1H, ArH), 5.07 (q, J = 8.9 Hz, 4H, OCH2CF3). 13C-NMR (DMSO-d6) δ 171.7, 170.9, 156.3, 149.1, 141.7, 140.2, 132.9, 129.2, 129.1, 125.7, 125.4, 93.9, 86.8, 63.6, 63.4, 63.1, 62.8. HRMS (ESI): m/z calcd for C16H11F6IN4O4 [M + Na]+: 586.9627, found 586.9603.
N-((4,6-bis(2,2,2-Trifluoroethoxy)pyrimidin-2-yl)carbamoyl)-4-chlorobenzamide (5). White solid, mp 162–163 °C, yield = 94%. 1H-NMR (DMSO-d6) δ 11.42 (s, 1H, NH), 11.22 (s, 1H, NH), 8.02 (d, J = 8.5 Hz, 2H, Ph), 7.60 (d, J = 8.6 Hz, 2H, Ph), 6.36 (s, 1H, ArH), 5.07 (q, J = 8.9 Hz, 4H, OCH2CF3). 13C-NMR (DMSO-d6) δ 170.8, 168.0, 156.4, 149.4, 139.1, 132.1, 131.1, 129.6, 125.6, 86.4, 63.6, 63.3, 63.0, 62.8. HRMS (ESI): m/z calcd for C16H11ClF6N4O4 [M + H]+: 473.0451, found 473.0442.
N-((4,6-bis(2,2,2-Trifluoroethoxy)pyrimidin-2-yl)carbamoyl)-2-(trifluoromethyl)benzamide (6). White solid, mp 51–53 °C, yield = 68%. 1H-NMR (DMSO-d6) δ 11.55 (s, 1H, NH), 10.91 (s, 1H, NH), 7.88 (d, J = 7.7 Hz, 1H, Ph), 7.85–7.71 (m, 3H, Ph), 6.40 (s, 1H, ArH), 5.07 (q, J = 8.9 Hz, 4H, OCH2CF3). 13C-NMR (DMSO-d6) δ 170.8, 170.0, 156.3, 149.0, 134.8, 133.5, 131.9, 129.3, 127.4, 127.1, 126.8, 126.5, 126.3, 125.6, 123.4, 86.7, 63.6, 63.3, 63.0, 62.8. HRMS (ESI): m/z calcd for C17H11F9N4O4 [M + H]+: 507.0715, found 507.0711.
N-((4,6-bis(2,2,2-Trifluoroethoxy)pyrimidin-2-yl)carbamoyl)-4-(trifluoromethyl)benzamide (7). White solid, mp 167–169 °C, yield = 93%. 1H-NMR (DMSO-d6) δ 11.61 (s, 1H, NH), 11.15 (s, 1H, NH), 8.17 (d, J = 8.1 Hz, 2H, Ph), 7.90 (d, J = 8.1 Hz, 2H, Ph), 6.38 (s, 1H, ArH), 5.07 (q, J = 8.9 Hz, 4H, OCH2CF3). 13C-NMR (DMSO-d6) δ 170.9, 167.9, 156.4, 149.3, 137.4, 133.7, 133.5, 130.1, 126.5, 126.5, 125.8, 125.6, 86.5, 63.7, 63.4, 63.1, 62.8. HRMS (ESI): m/z calcd for C17H11F9N4O4 [M + H]+: 507.0715, found 507.0713.
N-((4,6-bis(2,2,2-Trifluoroethoxy)pyrimidin-2-yl)carbamoyl)-5-chloro-2-methoxybenzamide (8). White solid, mp 164–166 °C, yield = 95%. 1H-NMR (DMSO-d6) δ 11.04 (s, 1H, NH), 10.98 (s, 1H, NH), 7.65 (d, J = 2.7 Hz, 1H, Ph), 7.61 (dd, J = 8.9, 2.8 Hz, 1H, Ph), 7.23 (d, J = 8.9 Hz, 1H, Ph), 6.39 (s, 1H, ArH), 5.06 (q, J = 8.9 Hz, 4H, OCH2CF3), 3.89 (s, 3H, CH3). 13C-NMR (DMSO-d6) δ 170.9, 167.3, 156.6, 156.3, 148.9, 133.9, 130.1, 125.4, 125.1, 115.3, 86.7, 63.5, 63.3, 63.0, 62.7, 57.5. HRMS (ESI): m/z calcd for C17H13ClF6N4O5 [M + H]+: 503.0557, found 503.0535.
N-((4,6-bis(2,2,2-Trifluoroethoxy)pyrimidin-2-yl)carbamoyl)-2,4-dichlorobenzamide (9). White solid, mp 109–110 °C, yield = 92%. 1H-NMR (DMSO-d6) δ 11.49 (s, 1H, NH), 10.88 (s, 1H, NH), 7.79 (d, J = 1.9 Hz, 1H, Ph), 7.68 (d, J = 8.2 Hz, 1H, Ph), 7.58 (dd, J = 8.2, 2.0 Hz, 1H, Ph), 6.40 (s, 1H, ArH), 5.06 (q, J = 8.9 Hz, 4H, OCH2CF3). 13C-NMR (DMSO-d6) δ 170.8, 168.3, 156.3, 149.0, 136.9, 134.6, 132.0, 131.4, 130.3, 128.5, 125.6, 86.8, 63.6, 63.3, 63.0. HRMS (ESI): m/z calcd for C16H10Cl2F6N4O4 [M + Na]+: 528.9881, found 528.9866.
N-((4,6-bis(2,2,2-Trifluoroethoxy)pyrimidin-2-yl)carbamoyl)-2,6-dichlorobenzamide (10). White solid, mp 62–64 °C, yield = 81%. 1H-NMR (DMSO-d6) δ 11.68 (s, 1H, NH), 10.80 (s, 1H, NH), 7.82–7.39 (m, 3H, Ph), 6.41 (s, 1H, ArH), 5.08 (q, J = 8.9 Hz, 4H, OCH2CF3). 13C-NMR (DMSO-d6) δ 170.9, 156.2, 148.8, 131.4, 129.2, 127.8, 125.6, 123.4, 121.2, 100.5, 86.9, 63.7, 63.4, 63.1, 62.9. HRMS (ESI): m/z calcd for C16H10Cl2F6N4O4 [M + Na]+: 528.9881, found 528.9866.
2-Chloro-N-((4,6-dimethylpyrimidin-2-yl)carbamoyl)benzamide (11). White solid, mp 186–188 °C, yield = 87%. 1H-NMR (DMSO-d6) δ 12.51 (s, 1H, NH), 10.61 (s, 1H, NH), 7.64 (dd, J = 7.6, 1.6 Hz, 1H, Ph), 7.61–7.52 (m, 2H, Ph), 7.47 (td, J = 7.3, 1.5 Hz, 1H, Ph), 6.98 (s, 1H, ArH), 2.33 (s, 6H, Ar-CH3). 13C-NMR (DMSO-d6) δ 168.8, 167.5, 157.6, 149.8, 136.3, 132.9, 130.8, 130.6, 129.8, 128.3, 116.0, 24.2. HRMS (ESI): m/z calcd for C14H13ClN4O2 [M + Na]+: 327.0625, found 327.0629.
2-Chloro-N-((4,6-dimethoxypyrimidin-2-yl)carbamoyl)benzamide (12). White solid, mp 155–157 °C, yield = 89%. 1H-NMR (DMSO-d6) δ 11.87 (s, 1H, NH), 10.67 (s, 1H, NH), 7.62 (dd, J = 7.6, 1.6 Hz, 1H, Ph), 7.59–7.50 (m, 2H, Ph), 7.46 (td, J = 7.3, 1.5 Hz, 1H, Ph), 5.97 (s, 1H, ArH), 3.82 (s, 6H, Ar-OCH3). 13C-NMR (DMSO-d6) δ 172.4, 168.4, 156.9, 149.4, 136.3, 132.8, 130.6, 130.5, 129.7, 128.3, 85.1, 55.2. HRMS (ESI): m/z calcd for C14H13ClN4O4 [M + Na]+: 359.0523, found 359.0524.
2-Chloro-N-((4,6-diethoxypyrimidin-2-yl)carbamoyl)benzamide (13). White solid, mp 113–115 °C, yield = 91%. 1H-NMR (DMSO-d6) δ 11.92 (s, 1H, NH), 10.61 (s, 1H, NH), 7.65 (dd, J = 7.6, 1.5 Hz, 1H, Ph), 7.57 (qd, J = 8.1, 4.1 Hz, 2H, Ph), 7.52–7.44 (m, 1H, Ph), 5.87 (s, 1H, ArH), 4.20 (q, J = 7.0 Hz, 4H, OCH2CH3), 1.26 (t, J = 7.1 Hz, 6H, OCH2CH3). 13C-NMR (DMSO-d6) δ 171.9, 167.9, 156.9, 149.4, 136.3, 135.8, 132.8, 130.6, 129.7, 128.3, 85.3, 64.5, 63.6, 15.2, 15.0. HRMS (ESI): m/z calcd for C16H17ClN4O4 [M + Na]+: 387.0836, found 387.0851.
2-Chloro-N-((4,6-dichloropyrimidin-2-yl)carbamoyl)benzamide (14). White solid, mp 156–158 °C, yield = 88%. 1H-NMR (DMSO-d6) δ 11.52 (s, 1H, NH), 11.24 (s, 1H, NH), 7.69 (s, 1H, ArH), 7.65 (dd, J = 7.6, 1.5 Hz, 1H, Ph), 7.57 (qd, J = 8.1, 1.7 Hz, 2H, Ph), 7.49 (td, J = 7.2, 1.9 Hz, 1H, Ph). 13C-NMR (DMSO-d6) δ 169.0, 162.5, 157.4, 148.8, 133.1, 130.8, 130.8, 130.0, 128.3, 117.0. HRMS (ESI): m/z calcd for C12H7Cl3N4O2 [M + Na]+: 366.9532, found 366.9549.
2-Chloro-N-((4-chloro-6-methylpyrimidin-2-yl)carbamoyl)benzamide (15). White solid, mp 126–128 °C, yield = 93%. 1H-NMR (DMSO-d6) δ 11.89 (s, 1H, NH), 10.98 (s, 1H, NH), 7.65 (dd, J = 7.6, 1.6 Hz, 1H, Ph), 7.63–7.51 (m, 2H, Ph), 7.47 (td, J = 7.3, 1.5 Hz, 1H, Ph), 7.32 (s, 1H, ArH), 2.40 (s, 3H, CH3). 13C-NMR (DMSO-d6) δ 171.4, 169.1, 168.3, 161.4, 157.5, 149.2, 138.1, 131.5, 130.5, 129.6, 127.9, 116.4, 24.2. HRMS (ESI): m/z calcd for C13H10Cl2N4O2 [M + Na]+: 347.0079, found 347.0064.
2-Chloro-N-((4-chloro-6-methoxypyrimidin-2-yl)carbamoyl)benzamide (16). White solid, mp 166–167 °C, yield = 87%. 1H-NMR (DMSO-d6) δ 11.64 (s, 1H, NH), 10.98 (s, 1H, NH), 7.65 (d, J = 7.5 Hz, 1H, Ph), 7.56 (d, J = 8.5 Hz, 2H, Ph), 7.48 (t, J = 7.3 Hz, 1H, Ph), 6.82 (s, 1H, ArH), 3.91 (s, 3H, OCH3). 13C-NMR (DMSO-d6) δ 171.9, 168.6, 160.8, 157.2, 149.0, 135.7, 132.9, 130.6, 129.9, 128.2, 102.6, 55.7. HRMS (ESI): m/z calcd for C13H10Cl2N4O3 [M + Na]+: 363.0028, found 363.0013.
2-Chloro-N-((4-chloro-6-(2,2,2-trifluoroethoxy)pyrimidin-2-yl)carbamoyl)benzamide (17). White solid, mp 144–146 °C, yield = 88%. 1H-NMR (DMSO-d6) δ 11.51 (s, 1H, NH), 11.07 (s, 1H, NH), 7.65 (dd, J = 7.5, 1.5 Hz, 1H, Ph), 7.62–7.51 (m, 2H, Ph), 7.48 (td, J = 7.3, 1.7 Hz, 1H, Ph), 7.09 (s, 1H, ArH), 5.10 (q, J = 8.8 Hz, 2H, OCH2CF3). 13C-NMR (DMSO-d6) δ 170.0, 169.1, 161.8, 156.9, 148.9, 135.6, 133.1, 130.8, 130.0, 128.2, 125.5, 123.3, 103.0, 63.8, 63.5, 63.2, 62.9. HRMS (ESI): m/z calcd for C14H9Cl2F3N4O3 [M + Na]+: 430.9901, found 430.9887.
2-Chloro-N-((4-chloro-6-morpholinopyrimidin-2-yl)carbamoyl)benzamide (18). White solid, mp 147–149 °C, yield = 85%. 1H-NMR (DMSO-d6) δ 11.78 (s, 1H, NH), 10.59 (s, 1H, NH), 7.62 (dd, J = 7.6, 1.6 Hz, 1H, Ph), 7.59–7.50 (m, 2H, Ph), 7.46 (td, J = 7.3, 1.5 Hz, 1H, Ph), 6.71 (s, 1H, ArH), 3.58 (dd, J = 23.7, 4.9 Hz, 8H, morpholine). 13C-NMR (DMSO-d6) δ 168.2, 163.6, 157.0, 149.3, 136.1, 132.9, 130.7, 130.6, 129.8, 128.3, 97.9, 66.6, 60.7. HRMS (ESI): m/z calcd for C16H15Cl2N5O3 [M + Na]+: 418.0450, found 418.0441.
2-Chloro-N-((4-chloro-6-(diethylamino)pyrimidin-2-yl)carbamoyl)benzamide (19). white solid, mp 151–153 °C, yield = 83%. 1H-NMR (DMSO-d6) δ 11.97 (s, 1H, NH), 10.46 (s, 1H, NH), 7.60 (dd, J = 7.6, 1.6 Hz, 1H, Ph), 7.58–7.49 (m, 2H, Ph), 7.45 (td, J = 7.3, 1.6 Hz, 1H, Ph), 6.49 (s, 1H, ArH), 3.44 (s, 4H, CH2CH3), 1.19–0.91 (m, 6H, CH2CH3). 13C-NMR (DMSO-d6) δ 168.1, 162.1, 159.4, 157.2, 149.6, 136.5, 132.7, 130.5, 130.5, 129.7, 128.3, 97.1, 43.3, 13.4. HRMS (ESI): m/z calcd for C16H17Cl2N5O2 [M + Na]+: 404.0657, found 404.0642.
2-Chloro-N-((4-methyl-6-(2,2,2-trifluoroethoxy)pyrimidin-2-yl)carbamoyl)benzamide (20). White solid, mp 56–58 °C, yield = 82%. 1H-NMR (DMSO-d6) δ 12.13 (s, 1H, NH), 10.79 (s, 1H, NH), 7.72–7.63 (m, 1H, Ph), 7.63–7.52 (m, 2H, Ph), 7.49 (td, J = 7.3, 1.6 Hz, 1H, Ph), 6.71 (s, 1H, ArH), 5.07 (q, J = 8.9 Hz, 2H, OCH2CF3), 2.34 (s, 3H, CH3). 13C-NMR (DMSO-d6) δ 171.4, 170.3, 169.3, 168.1, 161.4, 157.0, 149.4, 136.0, 132.9, 130.7, 129.9, 128.2, 102.1, 62.8, 62.5, 62.2, 61.9, 24.1. HRMS (ESI): m/z calcd for C15H12ClF3N4O3 [M + Na]+: 411.0448, found 411.0442.
2-Chloro-N-((4-(pyrrolidin-1-yl)-6-(2,2,2-trifluoroethoxy)pyrimidin-2-yl)carbamoyl)benzamide (21). White solid, mp 121–122 °C, yield = 81%. 1H-NMR (DMSO-d6) δ 12.55 (s, 1H, NH), 10.28 (s, 1H, NH), 7.61 (d, J = 7.4 Hz, 1H, Ph), 7.58–7.50 (m, 2H, Ph), 7.46 (td, J = 7.3, 1.7 Hz, 1H, Ph), 5.62 (s, 1H, ArH), 4.97 (q, J = 9.0 Hz, 2H, OCH2CF3), 3.25 (s, 2H, tetrahydropyrrole), 3.02 (s, 2H, tetrahydropyrrole), 1.97–1.82 (m, 2H, tetrahydropyrrole), 1.77–1.60 (m, 2H, tetrahydropyrrole). 13C-NMR (DMSO-d6) δ 168.7, 167.3, 161.8, 156.7, 149.8, 136.9, 132.5, 130.5, 130.3, 129.4, 128.3, 125.9, 81.1, 62.2, 61.9, 61.6, 47.3, 25.7. HRMS (ESI): m/z calcd for C18H17ClF3N5O3 [M + H]+: 444.1050, found 444.1047.
2-Chloro-N-((4-(hexylamino)-6-(2,2,2-trifluoroethoxy)pyrimidin-2-yl)carbamoyl)benzamide (22). White solid, mp 126–128 °C, yield = 83%. 1H-NMR (DMSO-d6) δ 12.40 (s, 1H, NH), 10.28 (s, 1H, NH), 7.85–7.36 (m, 5H, Ph), 5.62 (s, 1H, ArH), 4.93 (q, J = 9.0 Hz, 2H, OCH2CF3), 3.05 (s, 2H, N–CH2(CH2)4CH3), 1.46–1.07 (m, 8H, N–CH2(CH2)4CH3), 0.86 (t, J = 6.8 Hz, 3H, N(CH2)5CH3). 13C-NMR (DMSO-d6) δ 168.0, 165.1, 157.1, 151.8, 149.8, 136.7, 132.6, 130.5, 129.6, 128.3, 125.9, 123.6, 82.2, 62.1, 61.8, 41.3, 32.0, 29.6, 27.0, 23.1, 14.9. HRMS (ESI): m/z calcd for C20H23ClF3N5O3 [M + Na]+: 496.1339, found 496.1357.
2,4-Dichloro-N-((4,6-dimethylpyrimidin-2-yl)carbamoyl)benzamide (23). White solid, mp 185–186 °C, yield = 81%. 1H-NMR (DMSO-d6) δ 12.50 (s, 1H, NH), 10.60 (s, 1H, NH), 7.77 (d, J = 2.0 Hz, 1H, Ph), 7.67 (d, J = 8.2 Hz, 1H, Ph), 7.57 (dd, J = 8.2, 2.0 Hz, 1H, Ph), 6.99 (d, J = 2.7 Hz, 1H, ArH), 2.35 (s, 6H, CH3). 13C-NMR (DMSO-d6) δ 168.8, 167.0, 157.6, 149.8, 136.5, 135.4, 131.8, 131.1, 130.3, 128.5, 116.1, 24.3. HRMS (ESI): m/z calcd for C14H12Cl2N4O2 [M + Na]+: 361.0235, found 361.0208.
2,4-Dichloro-N-((4-methyl-6-(2,2,2-trifluoroethoxy)pyrimidin-2-yl)carbamoyl)benzamide (24). White solid, mp 155–157 °C, yield = 81%. 1H-NMR (DMSO-d6) δ 12.14 (s, 1H, NH), 10.77 (s, 1H, NH), 7.76 (d, J = 2.0 Hz, 1H, Ph), 7.70 (d, J = 8.3 Hz, 1H, Ph), 7.64–7.54 (m, 1H, Ph), 6.71 (s, 1H, ArH), 5.07 (q, J = 8.9 Hz, 2H, OCH2CF3), 2.37 (s, 3H, CH3). 13C-NMR (DMSO-d6) δ 170.4, 169.3, 167.5, 157.1, 149.5, 136.7, 135.0, 132.0, 131.2, 130.3, 128.5, 125.7, 102.2, 62.9, 62.6, 62.3, 62.0, 24.2. HRMS (ESI): m/z calcd for C15H11Cl2F3N4O3 [M + Na]+: 445.0058, found 445.0064.
2,6-Dichloro-N-((4-chloro-6-(2,2,2-trifluoroethoxy)pyrimidin-2-yl)carbamoyl)benzamide (25). White solid, mp 125–126 °C, yield = 87%. 1H-NMR (-DMSO-d6) δ 11.64 (s, 1H, NH), 10.90 (s, 1H, NH), 7.59 (d, J = 8.9 Hz, 2H, Ph), 7.56–7.48 (m, 1H, Ph), 7.12 (s, 1H, ArH), 5.11 (q, J = 8.9 Hz, 2H, OCH2CF3). 13C-NMR (DMSO-d6) δ 170.0, 165.0, 161.8, 156.8, 148.7, 137.7, 132.1, 131.9, 129.0, 125.4, 103.2, 63.6, 63.3, 63.0. HRMS (ESI): m/z calcd for C14H8Cl3F3N4O3 [M + Na]+: 464.9512, found 464.9502.
2,6-Dichloro-N-((4-chloro-6-(diethylamino)pyrimidin-2-yl)carbamoyl)benzamide (26). White solid, mp 169–171 °C, yield = 81%. 1H-NMR (DMSO-d6) δ 12.42 (s, 1H, NH), 10.44 (s, 1H, NH), 7.61–7.47 (m, 3H, Ph), 6.54 (s, 1H, ArH), 3.46 (s, 4H, CH2CH3), 1.12 (s, 6H, CH2CH3). 13C-NMR (DMSO-d6) δ 165.0, 164.0, 160.9, 158.5, 136.7, 131.1, 130.9, 128.1, 96.2, 42.6, 25.8, 12.4. HRMS (ESI): m/z calcd for C16H16Cl3N5O2 [M + H]+: 416.0448, found 416.0444.
2,6-Dichloro-N-((4-(diethylamino)-6-(2,2,2-trifluoroethoxy)pyrimidin-2-yl)carbamoyl)benzamide (27). White solid, mp 140–142 °C, yield = 87%. 1H-NMR (DMSO-d6) δ 12.84 (s, 1H, NH), 10.22 (s, 1H, NH), 8.60–6.40 (m, 3H, Ph), 5.84 (s, 1H, ArH), 4.96 (q, J = 9.0 Hz, 2H, OCH2CF3), 3.46 (s, 4H, CH2CH3), 1.12 (s, 6H, CH2CH3). 13C-NMR (DMSO-d6) δ 169.2, 168.2, 163.3, 156.7, 149.8, 138.1, 131.5, 130.6, 129.7, 127.9, 80.4, 62.6, 62.3, 62.0, 61.7, 43.2, 13.4. HRMS (ESI): m/z calcd for C18H18Cl2F3N5O3 [M + Na]+: 502.0636, found 502.0635.
N-((4-(Benzylamino)-6-(2,2,2-trifluoroethoxy)pyrimidin-2-yl)carbamoyl)-2,6-dichlorobenzamide (28). White solid, mp 76–78 °C, yield = 80%. 1H-NMR (DMSO-d6) δ 12.46 (d, J = 369.0 Hz, 1H, NH), 10.27 (s, 1H, NH), 8.25 (s, 1H, Ar-NH), 7.82–7.40 (m, 3H, Ph), 7.42–7.14 (m, 5H, Ph), 5.70 (s, 1H, ArH), 4.93 (q, J = 9.0 Hz, 2H, OCH2CF3), 4.39 (s, 2H, N–CH2Ph). 13C-NMR (DMSO-d6) δ 164.0, 155.9, 151.0, 148.6, 138.7, 136.7, 131.1, 130.8, 129.9, 128.3, 128.0, 127.9, 127.0, 126.1, 124.8, 122.6, 120.4, 81.6, 61.6, 61.3, 61.0, 60.7, 44.2, 30.6, 25.7. HRMS (ESI): m/z calcd for C21H16Cl2F3N5O3 [M + Na]+: 536.0480, found 536.0485.
N-((4-(Benzyl(ethyl)amino)-6-(2,2,2-trifluoroethoxy)pyrimidin-2-yl)carbamoyl)-2,6-dichlorobenzamide (29). White solid, mp 61–62 °C, yield = 75%. 1H-NMR (DMSO-d6) δ 12.72 (s, 1H, NH), 10.28 (s, 1H, NH), 7.71–7.08 (m, 8H, Ph), 5.85 (s, 1H, ArH), 4.95 (d, J = 9.0 Hz, 2H, OCH2CF3), 4.74 (s, 2H, N–CH2Ph), 3.41 (d, J = 52.6 Hz, 2H, CH2CH3), 1.08 (s, 3H, CH2CH3). 13C-NMR (DMSO-d6) δ 169.3, 167.5, 166.0, 165.2, 164.7, 156.6, 156.3, 133.7, 131.1, 129.8, 129.5, 129.0, 128.9, 128.1, 125.8, 123.6, 81.1, 62.5, 62.2, 61.9, 60.7, 30.9, 15.0. HRMS (ESI): m/z calcd for C23H20Cl2F3N5O3 [M + H]+: 542.0974, found 542.0971.
N-((5-(Sec-butyl)-4-chloro-6-(2,2,2-trifluoroethoxy)pyrimidin-2-yl)carbamoyl)-2,6-dichlorobenzamide (30). White solid, mp 37–38 °C, yield = 73%. 1H-NMR (DMSO-d6) δ 11.64 (s, 1H, NH), 10.81 (s, 1H, NH), 7.76–7.39 (m, 3H, Ph), 5.16 (q, J = 8.8 Hz, 2H, OCH2CF3), 3.21 (dt, J = 9.2, 6.8 Hz, 1H, CH), 1.90–1.74 (m, 1H, CHCH2CH3), 1.68 (dt, J = 13.8, 6.7 Hz, 1H, CHCH2CH3), 1.29 (d, J = 7.1 Hz, 3H, CHCH3), 0.83 (t, J = 7.4 Hz, 3H, CHCH2CH3). 13C-NMR (DMSO-d6) δ 167.7, 166.7, 160.4, 154.0, 148.7, 131.2, 129.1, 125.6, 123.4, 118.3, 63.9, 63.6, 63.3, 63.0, 31.6, 27.4, 18.6, 13.2. HRMS (ESI): m/z calcd for C18H16Cl3F3N4O3 [M + Na]+: 521.0138, found 521.0157.
3.10. Synthesis of N-((4,6-bis(2,2,2-Trifluoroethoxy)pyrimidin-2-yl)carbamothioyl)-2-chlorobenzamide (31)
Compound 31 was prepared by the method given in [22]. 2-Chlorobenzoic acid (5 mmol) and thionyl chloride (10 mL) were added to a 100 mL three-necked flask under N2. The reaction mixture was refluxed for 3 h. The excess thionyl chloride was evaporated under reduced pressure to give a colorless transparent liquid. Anhydrous acetonitrile (5 mL) was added to the residue, then KSCN (5 mmol) was added to the system and reacted at 80 °C for 1 h. The reaction mixture was cooled to the room temperature, filtered and neutralized with triethylamine. Then intermediate Ia (5 mmol) and TBAB (10 mmol) were added into reaction mixture. The mixture was heated to reflux for 4 h. When the reaction was completed, the reaction mixture was cooled to room temperature, diluted with 20 mL of EtOAc, washed twice with brine (20 mL), dried with anhydrous Na2SO4 and evaporated in vacuo. Finally, the residue was purified by silica gel column chromatography to afford compound 31 as a yellow solid, mp 93–95 °C, yield = 53%. 1H-NMR (DMSO-d6) δ 12.84 (s, 1H, NH), 12.45 (s, 1H, NH), 7.71–7.62 (m, 1H, Ph), 7.56 (dd, J = 6.5, 1.7 Hz, 2H, Ph), 7.51–7.42 (m, 1H, Ph), 6.51 (s, 1H, ArH), 5.11 (q, J = 8.9 Hz, 4H, OCH2CF3). 13C-NMR (DMSO-d6) δ 178.3, 170.8, 168.3, 156.2, 135.2, 133.3, 131.1, 130.7, 130.3, 128.2, 125.6, 123.4, 88.2, 63.9, 63.6, 63.3, 63.0. HRMS (ESI): m/z calcd for C16H11ClF6N4O3S [M + Na]+: 511.0042, found 511.0015.
3.11. Synthesis of 2,6-Dichloro-N-((4-(diethylamino)-6-(2,2,2-trifluoroethoxy)pyrimidin-2-yl)carbamothioyl)-benzamide (32a)
Compound 32a was synthesized by a method similar to that used for compound 31. Yellow solid, mp 42–43 °C, yield = 59%. 1H-NMR (DMSO-d6) δ 12.84 (s, 1H, NH), 10.22 (s, 1H, NH), 7.93–7.14 (m, 3H, Ph), 5.84 (s, 1H, ArH), 4.96 (q, J = 9.0 Hz, 2H, OCH2CF3), 3.46 (s, 4H, CH2CH3), 1.12 (s, 6H, CH2CH3). 13C-NMR (DMSO-d6) δ 169.2, 168.2, 163.2, 156.7, 149.8, 132.5, 131.5, 130.6, 129.7, 127.9, 80.4, 62.6, 62.3, 62.0, 61.7, 43.2, 13.4. HRMS (ESI): m/z calcd for C18H18Cl2F3N5O2S [M + Na]+: 518.0408, found 518.0407.
3.12. Synthesis of 2,6-dichloro-N-(N-(4-(Diethylamino)-6-(2,2,2-trifluoroethoxy)pyrimidin-2-yl)-carbamimidoyl)benzamide (32)
Compound 32 was synthesized by the method described in [23]. A solution of compound 32a (0.37 g, 0.75 mmol) and EDCI (0.28 g, 1.49 mmol) was stirred at 0 °C for 30 min and hexamethyldisilazide (HMDS, 1.2 g, 7.4 mmol) was added. After the mixture was stirred for 3 h under 0 °C, the mixture was raised to room temperature and stood for 24 h. The organic layer was diluted with EtOAc (20 mL), washed twice with a saturated NaHCO3 solution (20 mL) and brine (20 mL), dried with anhydrous Na2SO4 and evaporated in vacuo. Finally, the residue was purified by silica gel column chromatography to afford compound 32 as a white solid, mp 166–168 °C, yield = 81%. 1H-NMR (DMSO-d6) δ 11.25 (s, 1H, NH), 9.56 (s, 1H, NH), 9.48 (s, 1H, NH), 7.46 (d, J = 8.1 Hz, 2H, Ph), 7.35 (dd, J = 8.8, 7.5 Hz, 1H, Ph), 5.85 (s, 1H, ArH), 4.98 (q, J = 9.0 Hz, 2H, OCH2CF3), 3.69–3.40 (m, 4H, CH2CH3), 1.13 (t, J = 7.0 Hz, 6H, CH2CH3). 13C-NMR (DMSO-d6) δ 176.3, 169.0, 163.2, 160.1, 157.5, 140.6, 131.0, 130.6, 129.0, 128.9, 126.0, 123.7, 80.8, 62.7, 62.4, 62.2, 43.5, 13.6. HRMS (ESI): m/z calcd for C18H19Cl2F3N6O2 [M + Na]+: 501.0796, found 501.0791.
3.13. Synthesis of the Target Compounds 33–38
Compounds 33–38 were prepared by a method similar to that used for compounds 1–30.
5-Chloro-N-((4-chloro-6-(diethylamino)pyrimidin-2-yl)carbamoyl)-1-methyl-1H-pyrazole-4-carboxamide (33). White solid, mp 96–97 °C, yield = 88%. 1H-NMR (DMSO-d6) δ 11.27 (s, 1H, NH), 10.73 (s, 1H, NH), 8.30 (s, 1H, ArH), 6.50 (s, 1H, ArH), 3.86 (s, 3H, N–CH3), 3.43 (s, 4H, CH2CH3), 1.11 (t, J = 7.0 Hz, 6H, CH2CH3). 13C-NMR (DMSO-d6) δ 162.7, 162.0, 159.0, 157.1, 149.7, 140.2, 131.8, 113.0, 97.3, 42.9, 37.6, 13.6. HRMS (ESI): m/z calcd for C14H17Cl2N7O2 [M + H]+: 386.0899, found 386.0929.
N-((5-(sec-Butyl)-4-chloro-6-(diethylamino)pyrimidin-2-yl)carbamoyl)-5-chloro-1-methyl-1H-pyrazole-4-carboxamide (34). White solid, mp 159–161 °C, yield = 75%. 1H-NMR (DMSO-d6) δ 11.10 (s, 1H, NH), 10.87 (s, 1H, NH), 8.34 (s, 1H, ArH), 3.86 (s, 3H, N-CH3), 3.23 (dq, J = 14.0, 7.0 Hz, 2H, CHCH2CH3), 2.79 (q, J = 7.3 Hz, 1H, CH), 1.78 (dtd, J = 15.2, 13.6, 7.3 Hz, 2H, CH2CH3), 1.39 (d, J = 7.2 Hz, 3H, CHCH3), 1.22 (t, J = 2.8 Hz, 2H, CH2CH3), 1.15 (t, J = 7.0 Hz, 6H, CH2CH3), 0.73 (t, J = 7.4 Hz, 3H, CHCH2CH3). 13C-NMR (DMSO-d6) δ 169.4, 162.3, 158.9, 153.5, 149.7, 140.2, 131.9, 119.1, 112.7, 45.7, 37.5, 35.2, 27.9, 18.4, 13.8, 13.4. HRMS (ESI): m/z calcd for C18H25Cl2N7O2 [M + Na]+: 464.1344, found 464.1363.
5-Chloro-N-((4-chloro-6-(2,2,2-trifluoroethoxy)pyrimidin-2-yl)carbamoyl)-1-methyl-1H-pyrazole-4-carboxamide (35). White solid, mp 181–183 °C, yield = 85%. 1H-NMR (DMSO-d6) δ 11.40 (s, 1H, NH), 11.10 (s, 1H, NH), 8.37 (s, 1H, ArH), 7.07 (s, 1H, ArH), 5.11 (q, J = 8.9 Hz, 2H, OCH2CF3), 3.86 (s, 3H, N–CH3). 13C-NMR (DMSO-d6) δ 169.9, 162.8, 161.8, 156.8, 149.4, 140.3, 132.2, 112.3, 102.9, 63.5, 63.2, 62.9, 37.5. HRMS (ESI): m/z calcd for C12H9Cl2F3N6O3 [M + H]+: 434.9963, found 434.9975.
N-((5-(sec-Butyl)-4,6-dichloropyrimidin-2-yl)carbamoyl)-2-chloronicotinamide (36). White solid, mp 139–140 °C, yield = 86%. 1H-NMR (DMSO-d6) δ 11.49 (s, 1H, NH), 10.99 (s, 1H, NH), 8.55 (dd, J = 4.9, 1.9 Hz, 1H, ArH), 8.09 (dd, J = 7.6, 1.9 Hz, 1H, ArH), 7.58 (dd, J = 7.6, 4.8 Hz, 1H, ArH), 3.40 (dt, J = 9.0, 7.1 Hz, 1H, CH), 2.01–1.85 (m, 1H, CHCH2CH3), 1.73 (dt, J = 13.9, 7.1 Hz, 1H, CHCH2CH3), 1.33 (d, J = 7.2 Hz, 3H, CHCH3), 0.81 (t, J = 7.4 Hz, 3H, CHCH2CH3). 13C-NMR (DMSO-d6) δ 167.9, 154.5, 152.1, 148.9, 146.8, 139.1, 132.4, 129.8, 124.0, 37.1, 27.1, 18.2, 13.3. HRMS (ESI): m/z calcd for C15H14Cl3N5O2 [M + Na]+: 424.0111, found 424.0112.
2-Chloro-N-((4-(diethylamino)-6-(2,2,2-trifluoroethoxy)pyrimidin-2-yl)carbamoyl)nicotinamide (37). White solid, mp 124–126 °C, yield = 85%. 1H-NMR (DMSO-d6) δ 12.33 (s, 1H, NH), 10.30 (s, 1H, NH), 8.53 (dd, J = 4.9, 1.9 Hz, 1H, ArH), 8.06 (dd, J = 7.6, 1.9 Hz, 1H, ArH), 7.55 (dd, J = 7.6, 4.9 Hz, 1H, ArH), 5.84 (s, 1H, ArH), 4.97 (q, J = 9.0 Hz, 2H, OCH2CF3), 3.47 (s, 4H, CH2CH3), 1.11 (t, J = 7.0 Hz, 6H, CH2CH3). 13C-NMR (DMSO-d6) δ 169.2, 167.3, 163.1, 156.7, 151.5, 146.5, 138.6, 133.5, 125.9, 124.0, 80.5, 62.6, 62.3, 62.1, 61.8, 43.4, 13.5. HRMS (ESI): m/z calcd for C17H18ClF3N6O3 [M + Na]+: 469.0979, found 469.0966.
1-(4-Chloro-6-(diethylamino)pyrimidin-2-yl)-3-(2,6-dichlorophenyl)urea (38). White solid, mp 98–99 °C, yield = 78%. 1H-NMR (DMSO-d6) δ 10.90 (s, 1H, NH), 10.11 (s, 1H, NH), 7.58 (d, J = 8.1 Hz, 2H, Ph), 7.37 (t, J = 8.1 Hz, 1H, Ph), 6.47 (s, 1H, ArH), 3.33 (s, 4H, CH2CH3), 2.50 (s, 6H, CH2CH3). 13C-NMR (DMSO-d6) δ 161.3, 158.9, 157.5, 151.8, 133.9, 132.8, 129.5, 129.0, 95.6, 43.1, 12.8. HRMS (ESI): m/z calcd for C15H16Cl3N5O [M + H]+: 388.0499, found 388.0488.
3.14. Insecticidal Biological Assay
All bioassays were performed on representative test organisms reared in the laboratory. The bioassay was repeated at 25 ± 1 °C according to statistical requirements. Assessments were made on a dead/alive basis and mortality rates were corrected using Abbott’s formula [24]. Evaluations are based on a percentage scale of 0–100 in which 0 = no activity and 100 = total kill.
3.14.1. Toxicity against Mosquito (Culex pipiens pallens)
The toxicities of compounds 1–25, 31 and 37 against mosquito were evaluated according to the reported procedure [25,26]. One milliliter of different concentrated dilutions of each compound was added to 99 mL of water to obtain different concentrations of tested solution. Then 20 fourth-instar mosquito larvae were put into the solution. Percentage mortalities were evaluated 8 day after treatment. For comparative purposes, fipronil was tested under the same conditions, and each test was performed in triplicate.
3.14.2. Stomach Toxicity against Oriental Armyworm (Mythimna separata)
The stomach toxicities of compounds 1–25, 31, 37 and the contrast fipronil against oriental armyworm were evaluated by foliar application using the reported procedure [27]. For the foliar armyworm tests, individual corn leaves were placed on moistened pieces of filter paper in Petri dishes. The leaves were then sprayed with the test solution and allowed to dry. The dishes were infested with 10 fourth-instar Oriental armyworm larvae. Percentage mortalities were evaluated 3 days after treatment. Each treatment was performed three times
3.15. In Vitro Antifungal Bioassay
The antifungal activities were screened and evaluated by the poison plate technique [28]. All final compounds were dissolved in DMF (0.1 mL) before mixing with potato dextrose agar (PDA; 9.9 mL). The compounds were tested at a concentration of 50 μg mL−1. All fungi were cultivated in PDA at 27 ± 1 °C for 4 days to make new mycelium for the identification of antifungal activity. Then, mycelia dishes of approximately 5 mm diameter were cut from the culture medium. A mycelium was obtained using a germ-free inoculation needle and inoculated in the middle of the PDA plate aseptically. The inoculated plates were incubated at 27 ± 1 °C for 5 days. DMF in sterile distilled water served as the negative control, whereas hymexazol served as the positive control. Each treatment condition consisted of three replicates. Radial growth of the fungal colonies was measured, and the data were statistically analyzed. Inhibitory effects of the test compounds in vitro on these fungi were calculated by the formula I (%) = [(C − T)/(C − 0.5)] × 100, where C represents the diameter of fungal growth on untreated PDA, T represents the diameter of fungi on treated PDA, and I represents the inhibition rate.
3.16. In Vivo Antifungal Bioassay against Sclerotinia sclerotiorum
The requisite amounts of compounds 19 and 25 was disolved in sterile Tween 80 (0.1%, v/v) solution to give different concentration test solution (500, 1500 and 3000 μg mL−1). For protective activity assay [29], fresh leaves were sprayed with these solution (10 mL for each leave) until liquid flowed on surface at 24 h before inoculation. A colonized mycelial plug (5 mm in diameter) from a 5-day-old PDA culture of carbendazim-resistant Sclerotinia sclerotiorum was placed on the surface of fresh leaves. Inoculated leaves were placed at 25 °C with 80% relative humidity for disease development. After 5 days, the average lesion diameter was determined by measuring each lesion in two perpendicular directions. The lengths of the long and short axes were averaged and disease control efficacy was calculated as follows: disease control efficacy (%) = (lesion diameter in the water control-lesion diameter in the treatment)/lesion diameter in the water control × 100 [30].
3.17. Zebrafish Embryo Toxicity Assay
Zebrafish wild-type AB (Danio rerio AB) was introduced from the National Zebrafish Center (Wuhan, China). The zebrafish for experiment was cultured by the five-layer zebrafish culture system of Beijing Aisheng Technology Development Co., Ltd. (Beijing, China) to the fifth generation. Animals were housed in feeding system (pH 6.5–7.5, oxygen content >85%, water temperture 28 °C, conductivity 500 ± 50 μs cm−1, Light-dark ratio 14 h/10 h). Natural mortality during feeding was less than 1%. Fish were fed twice a day with Artemia salina, the total weight of feeding was 5 ± 1% of zebrafish body weight. For this experiment, a sexually mature 8-month zebrafish were used. Males and females were maintained separately until the night before the spawning at a ratio of 1:2 and the light-dark ratio was controlled at 14 h/10 h. Embryos were obtained by naturally mate [31].
Toxicological analyses were based on approved standard OECD TG 236: Fish Embryo Toxicity (FET) Test [32]. 20 embryos were distributed in each of different concentrations of the compounds to be analyzed, a positive control (3,4-dichloroaniline), a negative control (water) and a solvent control (10% Hank’s solution). All of the treatments were cared at 26 °C ± 1 °C, light/dark cycle of 14/10 h. The entire experiment was carried out for 96 h. All of the data were analyzed by SPSS 19.0 (IBM Corporation, New York, NY, USA), LC50 was corrected taking into account control mortality with Abbott’s formula [15].
4. Conclusions
In summary, a series of novel benzoylpyrimidinylurea derivatives were designed and synthesized. Although we did not find any compounds which possessed both insecticidal and antifungal as efficient as we expected, compounds 7 and 25 still exhibited excellent larvicidal activity against mosquito (Culex pipiens pallens) and broad-spectrum antifungal activity against fourteen phytopathogenic fungi, respectively. High efficiency, low toxicity and environmentally friendly pesticides are consistent with the requirements of sustainable agricultural development. Therefore, compounds 7 and 25 with low toxicity to zebrafish will be potential lead compounds to develop green mosquitocides and broad-spectrum fungicides.
Supplementary Materials
The following are available online. Figures S1–S101.
Author Contributions
R.S. conceived and designed the experiments; P.C., X.S. and W.K. performed the experiments, analyzed the data; X.S. drafted the manuscript; R.S. revised the manuscript; Y.F. and H.Z. gave some suggestion to the experiment.; P.C and X.S contributed equally to this work. All authors have read and approved the final manuscript.
Funding
This work was financially supported by the National Science Foundation of China (No. 21462028), Startup Foundation for Outstanding Young Scientists of Hainan University (kyqd1640) and Hainan Province Natural Science Foundation of China (No. 20163040).
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors. |
Figure 1.
Chemical structures of benzoylurea compounds.
Figure 2.
Chemical structures of compounds with pyrimidine groups.
Figure 3.
Design strategies of the target benzoylpyrimidinylurea derivatives.
Scheme 1.
Synthetic route for substituted 2-aminopyrimidines Ia–c, Id–f and Ig–m.
Scheme 2.
Synthetic route for substituted 2-aminopyrimidines In, Io and Ip.
Scheme 3.
Synthetic route for compounds 1–10.
Scheme 4.
Synthetic route for compounds 11–30.
Scheme 5.
Synthetic route for compounds 31 and 32.
Scheme 6.
Synthetic route for compounds 33–38.
Table 1.
Larvicidal Activities against Oriental Armyworm and Mosquito of Compounds 1–25, 31, 37.
| Compd. | Larvicidal Activity (%) | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Oriental Armyworm (µg mL−1) | Mosquito (µg mL−1) | ||||||||||
| 600 | 200 | 100 | 10 | 5 | 2 | 1 | 0.5 | 0.25 | 0.1 | ||
| 1 | 45 | - a | - | 100 | 100 | 60 | - | - | - | - | |
| 2 | 5 | - | - | 100 | 100 | 100 | 100 | 100 | 20 | - | |
| 3 | 100 | 100 | 60 | 100 | 100 | 60 | - | - | - | - | |
| 4 | 70 | - | - | 50 | - | - | - | - | - | - | |
| 5 | 65 | - | - | 50 | - | - | - | - | - | - | |
| 6 | 15 | - | - | 100 | 100 | 20 | - | - | - | - | |
| 7 | 65 | - | - | 100 | 100 | 100 | 100 | 100 | 100 | 20 | |
| 8 | 10 | - | - | 20 | - | - | - | - | - | - | |
| 9 | 100 | 100 | 40 | 100 | 100 | 40 | - | - | - | - | |
| 10 | 40 | - | - | 100 | 20 | - | - | - | - | - | |
| 11 | 20 | - | - | 10 | - | - | - | - | - | - | |
| 12 | 5 | - | - | 20 | - | - | - | - | - | - | |
| 13 | 60 | - | - | 100 | 100 | 70 | - | - | - | - | |
| 14 | 50 | - | - | 100 | 100 | 60 | - | - | - | - | |
| 15 | 30 | - | - | 20 | - | - | - | - | - | - | |
| 16 | 5 | - | - | 100 | 0 | - | - | - | - | - | |
| 17 | 30 | - | - | 5 | - | - | - | - | - | - | |
| 18 | 35 | - | - | 70 | - | - | - | - | - | - | |
| 19 | 30 | - | - | 10 | - | - | - | - | - | - | |
| 20 | 10 | - | - | 80 | - | - | - | - | - | - | |
| 21 | 20 | - | - | 5 | - | - | - | - | - | - | |
| 22 | 30 | - | - | 65 | - | - | - | - | - | - | |
| 23 | 15 | - | - | 60 | - | - | - | - | - | - | |
| 24 | 20 | - | - | 65 | - | - | - | - | - | - | |
| 25 | 40 | - | - | 30 | - | - | - | - | - | - | |
| 31 | 100 | 20 | - | 100 | 100 | 100 | 50 | - | - | - | |
| 37 | 20 | - | - | 15 | - | - | - | - | - | - | |
| Fipronil | - | - | - | 100 | 100 | 100 | 100 | 100 | 100 | 100 | |
a—no test data.
Table 2.
Fungicidal Activity of Compounds 1–38 against Phytopathogenic Fungi at 50 µg mL−1.
| Compd. | Inhibition Rate (%) | |||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| A.Sa | F.G | P.I | P.C | S.S | B.C | R.S | F.C | C.H | P.P | R.C | B.M | W.A | F.M | |
| 1 | 16.7 | 3.4 | 5.9 | 17.2 | 10.7 | 7.7 | 6.2 | 11.6 | 16.7 | 26.8 | 11.6 | 16.3 | 12.5 | 27.3 |
| 2 | 16.7 | 44.8 | 35.3 | 41.9 | 62.5 | 34.6 | 35.8 | 41.9 | 46.7 | 91.1 | 89.5 | 55.1 | 47.5 | 57.6 |
| 3 | 55.6 | 56.0 | 57.1 | 36.8 | 53.5 | 44.8 | 38.5 | 53.6 | 47.8 | 62.8 | 94.9 | 63.3 | 55.6 | 50.0 |
| 4 | 44.4 | 68.0 | 57.1 | 31.6 | 41.9 | 37.9 | 30.8 | 50.0 | 47.8 | 81.4 | 93.2 | 76.7 | 63.0 | 45.5 |
| 5 | 33.3 | 16.0 | 7.1 | 15.8 | 10.5 | 24.1 | 11.5 | 21.4 | 21.7 | 41.9 | 69.5 | 16.7 | 22.2 | 13.6 |
| 6 | 16.7 | 37.9 | 23.5 | 38.7 | 44.6 | 34.6 | 35.8 | 34.9 | 43.3 | 57.1 | 83.7 | 51.0 | 45.0 | 48.5 |
| 7 | 12.5 | 10.3 | 23.5 | 25.8 | 10.7 | 11.5 | 12.3 | 16.3 | 30.0 | 44.6 | 23.3 | 16.3 | 22.5 | 21.2 |
| 8 | 16.7 | 41.4 | 17.6 | 32.3 | 14.3 | 11.5 | 6.2 | 14.0 | 16.7 | 26.8 | 17.4 | 14.3 | 27.5 | 18.2 |
| 9 | 33.3 | 40.0 | 28.6 | 31.6 | 53.5 | 27.6 | 32.7 | 39.3 | 47.8 | 76.7 | 88.1 | 70.0 | 37.0 | 50.0 |
| 10 | 55.6 | 56.0 | 50.0 | 31.6 | 58.1 | 51.7 | 32.7 | 57.1 | 52.2 | 58.1 | 94.9 | 66.7 | 51.9 | 68.2 |
| 11 | 44.4 | 24.0 | 10.7 | 10.5 | 11.6 | 27.6 | 17.3 | 21.4 | 17.4 | 51.2 | 67.8 | 20.0 | 25.9 | 13.6 |
| 12 | 44.4 | 36.0 | 14.3 | 26.3 | 18.6 | 13.8 | 9.6 | 25.0 | 30.4 | 46.5 | 62.7 | 33.3 | 33.3 | 13.6 |
| 13 | 38.9 | 28.0 | 28.6 | 26.3 | 16.3 | 27.6 | 19.2 | 25.0 | 30.4 | 97.7 | 69.5 | 40.0 | 48.1 | 22.7 |
| 14 | 44.4 | 24.0 | 28.6 | 31.6 | 20.9 | 58.6 | 7.7 | 14.3 | 43.5 | 60.5 | 71.2 | 30.0 | 22.2 | 18.2 |
| 15 | 33.3 | 36.0 | 21.4 | 68.4 | 16.3 | 44.8 | 11.5 | 10.7 | 17.4 | 51.2 | 67.8 | 20.0 | 25.9 | 13.6 |
| 16 | 27.8 | 20.0 | 21.4 | 26.3 | 14.0 | 10.3 | 11.5 | 21.4 | 17.4 | 55.8 | 67.8 | 20.0 | 33.3 | 18.2 |
| 17 | 38.9 | 16.0 | 21.4 | 26.3 | 10.5 | 20.7 | 21.2 | 17.9 | 30.4 | 32.6 | 47.5 | 20.0 | 33.3 | 13.6 |
| 18 | 33.3 | 32.0 | 7.1 | 15.8 | 11.6 | 20.7 | 9.6 | 14.3 | 30.4 | 51.2 | 62.7 | 20.0 | 33.3 | 22.7 |
| 19 | 66.7 | 64.0 | 64.3 | 73.7 | 83.7 | 75.9 | 46.2 | 60.7 | 60.9 | 88.0 | 93.2 | 70.0 | 66.7 | 63.6 |
| 20 | 55.6 | 36.0 | 42.9 | 26.3 | 14.0 | 10.3 | 11.5 | 39.3 | 47.8 | 76.7 | 84.7 | 66.7 | 59.3 | 50.0 |
| 21 | 33.3 | 31.6 | 36.4 | 27.8 | 19.6 | 47.6 | 16.0 | 37.1 | 40.0 | 44.9 | 87.3 | 50.0 | 44.4 | 50.0 |
| 22 | 22.2 | 31.6 | 18.2 | 19.4 | 53.6 | 23.8 | 33.3 | 37.1 | 30.0 | 51.0 | 87.3 | 44.4 | 29.6 | 46.2 |
| 23 | 27.8 | 12.0 | 14.3 | 26.3 | 10.5 | 10.3 | 9.6 | 7.1 | 26.1 | 67.4 | 59.3 | 3.3 | 37.0 | 18.2 |
| 24 | 11.1 | 12.0 | 10.7 | 26.3 | 11.6 | 17.2 | 9.6 | 25.0 | 30.4 | 67.4 | 71.2 | 13.3 | 14.8 | 9.1 |
| 25 | 55.6 | 44.0 | 57.1 | 52.6 | 90.7 | 89.7 | 57.7 | 60.7 | 69.6 | 84.2 | 94.9 | 66.7 | 59.3 | 63.6 |
| 26 | 44.4 | 31.6 | 54.5 | 61.1 | 82.1 | 47.6 | 30.9 | 48.6 | 60.0 | 51.0 | 92.7 | 55.6 | 48.1 | 53.8 |
| 27 | 22.2 | 31.6 | 27.3 | 19.4 | 26.8 | 23.8 | 18.5 | 34.3 | 35.0 | 65.3 | 89.1 | 47.2 | 40.7 | 53.8 |
| 28 | 16.7 | 18.4 | 27.3 | 27.8 | 53.6 | 14.3 | 6.2 | 14.3 | 5.0 | 6.1 | 58.2 | 22.2 | 14.8 | 26.9 |
| 29 | 11.1 | 5.3 | 4.5 | 27.8 | 17.9 | 19.0 | 6.2 | 31.4 | 25.0 | 26.5 | 72.7 | 27.8 | 14.8 | 30.8 |
| 30 | 31.3 | 45.5 | 21.1 | 27.3 | 33.3 | 12.5 | 37.9 | 16.7 | 45.5 | 56.4 | 66.2 | 28.9 | 21.4 | 27.3 |
| 31 | 8.3 | 15.8 | 20.7 | 24.0 | 5.4 | 31.0 | 6.6 | 8.3 | 9.5 | 13.2 | 27.3 | 29.6 | 7.4 | 17.9 |
| 32 | 11.1 | 21.1 | 27.3 | 22.2 | 23.2 | 14.3 | 6.2 | 17.1 | 15.0 | 8.2 | 56.4 | 19.4 | 18.5 | 30.8 |
| 33 | 31.3 | 27.3 | 31.6 | 40.9 | 29.4 | 18.8 | 22.7 | 36.7 | 50.0 | 89.7 | 78.4 | 44.7 | 21.4 | 40.9 |
| 34 | 43.8 | 31.8 | 15.8 | 4.5 | 33.3 | 18.8 | 18.2 | 10.0 | 27.3 | 66.7 | 20.3 | 31.6 | 14.3 | 18.2 |
| 35 | 12.5 | 27.3 | 15.8 | 4.5 | 11.8 | 18.8 | 12.1 | 6.7 | 18.2 | 46.2 | 37.8 | 15.8 | 10.7 | 18.2 |
| 36 | 37.5 | 36.4 | 31.6 | 27.3 | 49.0 | 31.3 | 30.3 | 56.7 | 50.0 | 79.5 | 78.4 | 68.4 | 57.1 | 54.5 |
| 37 | 44.4 | 44.7 | 50.0 | 55.6 | 81.7 | 47.6 | 34.6 | 54.3 | 40.0 | 22.4 | 89.1 | 61.1 | 59.3 | 65.4 |
| 38 | 44.4 | 47.4 | 50.0 | 55.6 | 37.5 | 71.4 | 67.9 | 65.7 | 65.0 | 42.9 | 98.2 | 63.9 | 70.4 | 61.5 |
| Pyrimethanil | 75.0 | 36.4 | 21.1 | 90.9 | 100 | 87.5 | 93.9 | 28.0 | 94.4 | 96.2 | 95.7 | 22.2 | 23.8 | 25.0 |
aA.S: Alternaria solani; F.G: Fusarium graminearum; P.I: Phytophthora infestans; S.S: Sclerotinia sclerotiorum; B.C: Botrytis cinereal; R.S: Rhizoctonia solani; F.O: Fusarium oxysporum f.sp.cucumerinum; C.A: Cercospora arachidicola; P.P: Physalospora piricola; R.C: Rhizoctonia cerealis; B.M: Bipolaris maydis; W.A: Watermelon-anthracnose; F.M: Fusarium moniliforme; P.C: Phytophthora capsica.
Table 3.
Inhibitory Effects of Compounds 19 and 25 against Phytopathogenic Fungi.
| Compd. | Phytopathogens | EC50 (μg mL−1) | Toxic Regression Eq | R b |
|---|---|---|---|---|
| 19 | S.Sa | 10.268 | y = −2.048 + 2.025x | 0.9757 |
| P.P | 15.891 | y = −3.842 + 3.198x | 0.9778 | |
| R.C | 14.744 | y = −4.221 + 3.612x | 0.9844 | |
| B.C | 17.175 | y = −1.600 + 1.295x | 0.9762 | |
| 25 | S.S | 7.072 | y = −1.436 + 1.691x | 0.9752 |
| P.P | 4.604 | y = −1.128 + 1.701x | 0.9597 | |
| R.C | 13.231 | y = −3.657 + 3.260x | 0.9695 | |
| B.C | 7.050 | y = −2.941 + 3.467x | 0.9618 |
aS.S: Sclerotinia sclerotiorum; B.C: Botrytis cinereal; P.P: Physalospora piricola; R.C: Rhizoctonia cerealis; b R: regression coefficient.
Table 4.
In vivo Protective Effects of Compounds 19 and 25 against Sclerotinia sclerotiorum.
| Compd. | Concn (μg mL−1) | Protection Effect (%) |
|---|---|---|
| 19 | 3000 | 55.7 |
| 1500 | 35.2 | |
| 500 | 34.1 | |
| 25 | 3000 | 83.0 |
| 1500 | 35.2 | |
| 500 | 20.5 | |
| Carbendazim | 500 | 100 |
Table 5.
The Zebrafish Embryo Acute Toxicity of Compounds 7 and 25.
| Compd. | Time (h) | LC50 (μg mL−1) | Toxic Regression Eq | R | 95% Confidence Limits |
|---|---|---|---|---|---|
| 7 | 96 | 378.387 | y = −4.378 + 1.698x | 0.9617 | 325.159–369.971 |
| 25 | 24 | 38.187 | y = −2.193 + 1.387x | 0.8718 | 23.899–67.569 |
| 48 | 30.435 | y = −2.142 + 1.444x | 0.9154 | 18.065–48.014 | |
| 72 | 26.886 | y = −2.274 + 1.591x | 0.9508 | 16.271–39.781 | |
| 96 | 21.668 | y = −2.131 + 1.595x | 0.9762 | 11.777–31.651 |
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Chen, P.; Song, X.; Fan, Y.; Kong, W.; Zhang, H.; Sun, R. Investigation of Novel Pesticides with Insecticidal and Antifungal Activities: Design, Synthesis and SAR Studies of Benzoylpyrimidinylurea Derivatives. Molecules 2018, 23, 2203. https://doi.org/10.3390/molecules23092203
AMA Style
Chen P, Song X, Fan Y, Kong W, Zhang H, Sun R. Investigation of Novel Pesticides with Insecticidal and Antifungal Activities: Design, Synthesis and SAR Studies of Benzoylpyrimidinylurea Derivatives. Molecules. 2018; 23(9):2203. https://doi.org/10.3390/molecules23092203
Chicago/Turabian StyleChen, Peiqi, Xiangmin Song, Yongmei Fan, Weihao Kong, Hao Zhang, and Ranfeng Sun. 2018. "Investigation of Novel Pesticides with Insecticidal and Antifungal Activities: Design, Synthesis and SAR Studies of Benzoylpyrimidinylurea Derivatives" Molecules 23, no. 9: 2203. https://doi.org/10.3390/molecules23092203
