Synthesis and Insecticidal Activity of Mesoionic Pyrido[1,2-α]pyrimidinone Derivatives Containing a Neonicotinoid Moiety

Mesoionic pyrido[1,2-α]pyrimidinone derivatives containing a neonicotinoid moiety were designed, synthesized, and evaluated for their insecticidal activity. Some of the title compounds showed remarkable insecticidal properties against Aphis craccivora. Compound I13 exhibited satisfactory insecticidal activity against A. craccivora. Meanwhile, label-free proteomics analysis of compound I13 treatment identified a total of 821 proteins. Of these, 35 proteins were up-regulated, whereas 108 proteins were down-regulated. Differential expressions of these proteins reflected a change in cellular structure and metabolism.


Introduction
Wide application of insecticides with the same mode of action has led to insect resistance; it is vitally important to develop novel insecticides with a new mode of action [1,2]. Mesoionic compounds are usually called non-benzenoid aromatics; these are polar and easily enter the hydrophilic cavity through the lipid barrier [3]. Besides these unique physical and chemical properties, they also possess various bioactivities, such as antifungal [4], anti-inflammatory [5][6][7], and analgesic activities [8][9][10][11]. They are inhibitors of cyclic AMP phosphodiesterase and antagonists of adenosine receptors [12,13], and show antibacterial [14][15][16], anti-tumor [17], and insecticidal [18][19][20][21][22] activities. Encouraged by these characteristics of mesoionic compounds, many researchers have studied the potential applications of mesoionics [23]. Recently, DuPont has discovered that Triflumezopyrim (Figure 1), a new commercial insecticide with a distinct mode of action and register in China in 2016, provides new insight into the application of mesoionic compounds in pesticides with high efficiency and environmentally friendly properties [19,24,25]. Mesoionic compounds in this field were also studied by DuPont [21]. Mesoionic compounds may be considered a novel pesticide.

Chemistry
As shown in Scheme 1, intermediates A, B, C, and the title compound I were prepared according to the reported methods. The structures of the title compounds were characterized by melting point, 1 H-NMR, 13 C-NMR, and HRMS. All copies of the spectrum for compounds I1-I28 are available in Supplementary Materials.

Chemistry
As shown in Scheme 1, intermediates A, B, C, and the title compound I were prepared according to the reported methods. The structures of the title compounds were characterized by melting point, 1 H-NMR, 13 C-NMR, and HRMS. All copies of the spectrum for compounds I1-I28 are available in Supplementary Materials.

Biological Evaluation
The insecticidal activities of the title compounds I1-I28 against A. craccivora were assayed. The commercial agent imidacloprid and triflumezopyrim were used as controls. The biassay showed that the title compounds exhibited moderate activities. Among the title compounds, I13 exhibited good insecticidal activities against A. craccivora with a mortality rate of 100% at 500 and 200 μg/mL, respectively, which was equal to those of imidacloprid (100%) and triflumezopyrim (100%). When the concentration of the compound I13 is reduced from 200 to 100 μg/mL, compound I13 still shows good mortality rate (92%) against A. craccivora. However, a great decrease of bioactivity (30%) was observed when the concentration was reduced from 100 to 50 μg/mL (Table 1). Meanwhile, at 500 μg/mL, compounds I1, I2, I6, I7, I8, I19, I20, I22, I23, I25, and I28 exhibited moderate activities (85%, 69%, 51%, 77%, 51%, 58%, 62%, 62%, 58%, 62%, and 62%, respectively) against A. craccivora. However, other compounds exhibited weak and inactivitive activities. Based on the above findings, when R2 was 2-Cl-pyridin-5-yl group, the substituents of phenyl ring R1 on the parent compound I affected the activity against A. craccivora. In short, the position of the substituents is a key factor, while the electron effect of the substituent is a secondary factor. The target compounds having a p-position substituent exhibit good activities (I7, I13, I19, and I23), and the target compounds having an o-position substituent exhibit weak activities (I3 and I9), while the target compounds having a m-position substituent show no activities (I5 and I15). Meanwhile, I25 and I27 with heterocycle moiety exhibited moderate activity. When R2 turns to 2-Cl-thiazol-5-yl group, the compounds Scheme 1. General synthetic procedure for the title compounds I1-I28.

Biological Evaluation
The insecticidal activities of the title compounds I1-I28 against A. craccivora were assayed. The commercial agent imidacloprid and triflumezopyrim were used as controls. The biassay showed that the title compounds exhibited moderate activities. Among the title compounds, I13 exhibited good insecticidal activities against A. craccivora with a mortality rate of 100% at 500 and 200 µg/mL, respectively, which was equal to those of imidacloprid (100%) and triflumezopyrim (100%). When the concentration of the compound I13 is reduced from 200 to 100 µg/mL, compound I13 still shows good mortality rate (92%) against A. craccivora. However, a great decrease of bioactivity (30%) was observed when the concentration was reduced from 100 to 50 µg/mL (Table 1). Meanwhile, at 500 µg/mL, compounds I1, I2, I6, I7, I8, I19, I20, I22, I23, I25, and I28 exhibited moderate activities (85%, 69%, 51%, 77%, 51%, 58%, 62%, 62%, 58%, 62%, and 62%, respectively) against A. craccivora. However, other compounds exhibited weak and inactivitive activities. Based on the above findings, when R 2 was 2-Cl-pyridin-5-yl group, the substituents of phenyl ring R 1 on the parent compound I affected the activity against A. craccivora. In short, the position of the substituents is a key factor, while the electron effect of the substituent is a secondary factor. The target compounds having a p-position substituent exhibit good activities (I7, I13, I19, and I23), and the target compounds having an o-position substituent exhibit weak activities (I3 and I9), while the target compounds having a m-position substituent show no activities (I5 and I15). Meanwhile, I25 and I27 with heterocycle moiety exhibited moderate activity. When R 2 turns to 2-Cl-thiazol-5-yl group, the compounds exhibit lower activity. Interestingly, when R 1 was benzyl with no substituents on the benzene ring, the corresponding compounds showed moderate insecticidal properties, such as I1 (mortality rate: 85%) and I2 (mortality rate: 69%). In a word, the structures R 1 and R 2 of title compounds were combined with the activity. Among them, compound I13 could offer considerable potential for further development as a new lead compound in modern drug discovery.  Table S1). As shown in Supplementary Materials Table S1, 678 proteins (82.6%) had non-specific expression, and 143 proteins were differentially expressed, out of which 35 proteins were up-regulated, whereas 108 proteins were down-regulated. Meanwhile, a volcanic map (Figure 3) was plotted to better understand the expression of this differential proteins (Supplementary Materials Table S2), which included 35 up-regulated proteins (red dots) and 108 down-regulated proteins (green dots). expression of this differential proteins (Supplementary Materials Table S2), which included 35 up-regulated proteins (red dots) and 108 down-regulated proteins (green dots).  Figure 4A shows the different expressions of proteins, which were grouped by biological process (BP). The up-regulated proteins were involved in protein folding and translation. The down-regulated proteins were mainly involved in DNA-templated transcription, protein folding, translation, regulation of translational initiation, cell redox homeostasis, and DNA repair. Grouped according to cellular components (CC), Figure 4B showed that integral component of membrane and ribosome showed decreased expression. The structural constituent of cuticle, structural constituent of ribosome, and actin binding were mapped to the up-regulated proteins of molecular function (MF) ( Figure 4C).

Bioinformatics Analysis
To study the potential link between differentially expressed protein and biological functions, we used KEGG database to identify potential pathways for differential proteins in the treatment groups. Protein processing in endoplasmic reticulum (pathway ID: ko04141) is the main enrichment pathway. The enrichment pathway includes a total of 10 specific proteins, such as 6 heat shock proteins (HSP), 2 protein disulfide-isomerase, 1 transitional endoplasmic reticulum ATPase TER94, and 1 DnaJ-lik protein. HSP are in relation to temperature stress and a family of proteins that are produced by cells in response to exposure to stressful conditions. Furthermore, among different expressions proteins, we found some proteins were connected with temperature stress ((2 cold-shock proteins (IDS: Q492L6 and A0A0M3RSL4) and 2 HSP proteins (IDS: A0A172JCK4 and A0A0H5BX82)). Literatures revealed that CSPs can bind mRNA and regulate ribosomal translation, mRNA degradation, and the rate of transcription termination [33,34]. CSP, which can inhibit cell division and reducing apoptosis, is widely involved in the replication, transcription, translatin, protein folding, and membrane fluidity of various genes at low temperatures and plays a significant role in the protection of organ tissue at low temperatures [35][36][37]. However, the effect of cold shock had more of a general nature, e.g., slowing down of metabolic activities. Recent observations have changed this outlook on cold-shock response and have shown it to be a specific response of a cell at various levels, such as cytoplasmic membrane, ribosomes, nucleic acids, and  Figure 4A shows the different expressions of proteins, which were grouped by biological process (BP). The up-regulated proteins were involved in protein folding and translation. The down-regulated proteins were mainly involved in DNA-templated transcription, protein folding, translation, regulation of translational initiation, cell redox homeostasis, and DNA repair. Grouped according to cellular components (CC), Figure 4B showed that integral component of membrane and ribosome showed decreased expression. The structural constituent of cuticle, structural constituent of ribosome, and actin binding were mapped to the up-regulated proteins of molecular function (MF) ( Figure 4C).

Bioinformatics Analysis
To study the potential link between differentially expressed protein and biological functions, we used KEGG database to identify potential pathways for differential proteins in the treatment groups. Protein processing in endoplasmic reticulum (pathway ID: ko04141) is the main enrichment pathway. The enrichment pathway includes a total of 10 specific proteins, such as 6 heat shock proteins (HSP), 2 protein disulfide-isomerase, 1 transitional endoplasmic reticulum ATPase TER94, and 1 DnaJ-lik protein. HSP are in relation to temperature stress and a family of proteins that are produced by cells in response to exposure to stressful conditions. Furthermore, among different expressions proteins, we found some proteins were connected with temperature stress ((2 cold-shock proteins (IDS: Q492L6 and A0A0M3RSL4) and 2 HSP proteins (IDS: A0A172JCK4 and A0A0H5BX82)). Literatures revealed that CSPs can bind mRNA and regulate ribosomal translation, mRNA degradation, and the rate of transcription termination [33,34]. CSP, which can inhibit cell division and reducing apoptosis, is widely involved in the replication, transcription, translatin, protein folding, and membrane fluidity of various genes at low temperatures and plays a significant role in the protection of organ tissue at low temperatures [35][36][37]. However, the effect of cold shock had more of a general nature, e.g., slowing down of metabolic activities. Recent observations have changed this outlook on cold-shock response and have shown it to be a specific response of a cell at various levels, such as cytoplasmic membrane, ribosomes, nucleic acids, and proteins. So, we hypothesized that compound I13 can change the sensitivity to temperature and then lead to the death of A. craccivora. Molecules 2018, 23, x 6 of 11

Synthesis
NMR spectra were recorded on a JEOL ECX-500 spectrometer (JEOL, Tokyo, Japan). High-resolution mass spectra (HRMS) were acquired in positive mode on a MALDI SYNAPT G2 high-definition mass spectrometer (Waters, Milford, MA, USA). Melting points were taken on a Büchi B-545 melting point apparatus (Büchi Labortechnik AG, Flawil, Switzerland, uncorrected). Silica gel GF254-coated glass plates (Branch Qingdao Haiyang Chemical Co., Qingdao, China) were used for thin layer chromatography (TLC) under detection at 254 nm. Silica gel 200-300 mesh (Branch Qingdao Haiyang Chemical Co., Qingdao, China) was applied to column chromatography. All chemical reagents were commercially available and used without further purification.

Synthesis
NMR spectra were recorded on a JEOL ECX-500 spectrometer (JEOL, Tokyo, Japan). High-resolution mass spectra (HRMS) were acquired in positive mode on a MALDI SYNAPT G2 high-definition mass spectrometer (Waters, Milford, MA, USA). Melting points were taken on a Büchi B-545 melting point apparatus (Büchi Labortechnik AG, Flawil, Switzerland, uncorrected). Silica gel GF 254 -coated glass plates (Branch Qingdao Haiyang Chemical Co., Qingdao, China) were used for thin layer chromatography (TLC) under detection at 254 nm. Silica gel 200-300 mesh (Branch Qingdao Haiyang Chemical Co., Qingdao, China) was applied to column chromatography. All chemical reagents were commercially available and used without further purification.

Biological Assay
Bioassays of insecticidal activity against A. craccivora were investigated via a slightly modified FAO dip test method [44,45]. Tender shoots of soybeans with adult aphids were dipped in diluted solutions of the title compounds containing Triton X-100 (0.1%) for 5 s. Excess liquid was removed, and the shoots were placed in the conditioned room (25 ± 1 • C, 50% RH). Triflumezopyrim and Imidacloprid were used as positive controls. Mortality rates were recorded after 24 h.

Sample Preparation
Tender shoots of soybeans with 50-100 adult aphids were dipped in 100 µg/mL of I13 solution (diluted by Triton X-100) for 5 s. Excess liquid was removed, and the shoots were placed in the conditioned room (25 ± 1 • C, 50% RH). The control groups were handled with 0.1% Triton X-100, and each treatment was repeated thrice. Samples of control and I13-treated insects were collected at 12 h after treatment and were frozen for protein extraction [46].

Proteins Extraction for LC−MS/MS Analysis
The total proteins of A. craccivora were extracted by a modified method [47,48]. First, samples of control and I13-treated insects were homogenized to fine powder (by mortar and pestle in liquid nitrogen). Ice-cold protein extraction buffer (0.5 M Tris-HCl (pH 7.5), 0.7 M sucrose, 0.1 M KCl, 50 mM EDTA, and 40 mM dithiothreitol (DTT)) lysed the total soluble protein at room temperature for 15 min. Then, after 30 min of shaking, extraction was conducted by an equal volume of Tris-phenol. Centrifugation was performed at 8000 g and 4 • C for 15 min (twice extraction). Five times volume of 0.1 M ammonium acetate in methanol was added to the collected supernatant, which was maintained at −20 • C overnight and then centrifuged at 8000 g for 10 min at 4 • C. Finally, the resulting pellets were washed by ice-cold acetone containing 1% (w/v) DTT thrice. After drying for 2 h in vacuum drier, the samples were dissolved in 100 µL of the rehydration solution (8 M (w/v) urea, 0.1 M (w/v) Tris, 10 mM dithiothreitol (DTT). Then, the concentration of total protein was determined using the Bradford method [49]. Before the LC-MS/MS analysis, protein was digested with trypsin using reported methods [50].

LC-MS/MS Analysis, Database Searching, and Bioinformatics Analysis
All samples were analyzed via the LC-MS/MS combined system (Nano LC-1DTM plus system (Eksigent, Dublin, CA), TripleTOF 5600 MS (Foster City, CA, USA)). First, a full loop injection was used for 8 µL peptide samples. They were desalted on a ChromXP Trap column (Nano LC TRAP Column, 3 µm C18-CL, 120 A, 350 µm × 0.5 mm, Foster City, CA, USA). Then, the eluted samples were placed into column-Nano LC C18 reversed-phase column (3C18-CL, 75 µm × 15 cm, Foster City, CA, USA) for a second analysis. Under the flow rate of 300 nL/min, a combination of mobile phases, i.e., A mobile phase (5% ACN, 0.1% FA) and B mobile phase (95% ACN, 0.1% FA), was eluted over 120 min. Analyst (R) Software (TF1.6) can automatically switch between TOF-MS and Product Ion acquisition by the data-dependent mode on TripleTOF 5600 MS.
MaxQuant version 1.5.2.8 (http://www.coxdocs.org/doku.php?id=maxquant:common: download_and_installation)was used to manipulate raw data. The proteome of aphids was downloaded from UniProt, which contained 68,023 proteins that were searched via Andromeda search engine [51,52]. To ensure that only significant peptides were accepted for the identification, the false discovery rate (FDR) was set to 0.01. The difference of expression between the control group and treatment group was compared by the label-free quantification with a minimum of two ratio counts to determine the normalized protein intensity. The differentially accumulated proteins between control and treatment groups were identified via a two-sample unpaired t-test. The iBAQ value was used for t-test. Proteins with ANOVA analysis of p value ≤ 0.05 were considered differentially expressed.
All differentially expressed proteins were annotated with all aphid proteins using the DAVID 6.8 (https://david.ncifcrf.gov/content.jsp?file=Contact.html) [53,54]. The Fisher's Exact Test (Fisher) exact test and FDR correction method [55][56][57] was used to identify the differentially expressed proteins based on GO (Gene Ontology, a gene function in a standardized classification system) categories in biological process (BP), cellular components (CC), and molecular functions (MF). The results are listed in Supplementary Materials Table S3. Some GO comments were listed after ranking according to the p-value sort. The smallest of the top 10 were shown in the column chart.

Conclusions
In summary, mesoionic pyrido[1,2-α]pyrimidinones derivatives containing a neonicotinoid moiety were designed, synthesized, and evaluated for their insecticidal activity. Results of bioassays indicated that these compounds displayed satisfactory insecticidal properties against A. craccivora. In particular, compound I13 showed 92% mortality at a concentration of 100 µg/mL. Using the label-free proteomics to analyze the differentially expressed proteins after compound I13 treatment, the differential expression of these proteins reflected the change in cellular structure and metabolism. Notably, these findings demonstrated that the synthesis of mesoionic pyrido[1,2-α]pyrimidinones derivatives containing a neonicotinoid moiety could be considered as a new template for pesticide development. These interesting bioactivities and responses of label-free quantitative proteomics led to further research by our group.