Synthesis and insecticidal activity of mesoionic pyrido[1,2-α]pyrimidinone derivatives containing a neonicotinoid moiety

Article abstract of DOI:10.3390/molecules23051217

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.

Full text of DOI:10.3390/molecules23051217

molecules
Article
Synthesis and Insecticidal Activity of Mesoionic
Pyrido[1,2-α]pyrimidinone Derivatives Containing
a Neonicotinoid Moiety
Jianke Pan, Lu Yu, Dengyue Liu and Deyu Hu *
State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key Laboratory of
Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou University, Huaxi District,
Guiyang 550025, China; jkpan_1@163.com (J.P.); yuji570@163.com (L.Y.); 18230826058@163.com (D.L.)
* Correspondence: dyhu@gzu.edu.cn; Tel.: +86-851-882-92170
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 12 March 2018; Accepted: 14 May 2018; Published: 19 May 2018
Abstract: 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.
Keywords: mesoionic; pyrido[1,2-α]pyrimidinones; neonicotinoid; insecticidal activity; proteomic
1. 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 [
are usually called non-benzenoid aromatics; these are polar and easily enter the hydrophilic cavity
through the lipid barrier [ ]. Besides these unique physical and chemical properties, they also possess
various bioactivities, such as antifungal [
They are inhibitors of cyclic AMP phosphodiesterase and antagonists of adenosine receptors [12
and show antibacterial [14 16], anti-tumor [17], and insecticidal [18
1,2]. Mesoionic compounds
3
4], anti-inflammatory [
5
–
7
], and analgesic activities [
8
–
11].
,13],
–
–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.
Neonicotinoids are widely used to prevent and control various diseases in plants, animals, and
humans [26]. They are the newest class of synthetic insecticides to emerge in the past two decades,
and they are also the best-selling insecticide [27]. Many neonicotinoid pesticides have been launched
to the market, including imidacloprid [28], nitenpyram [29], acetamiprid [30], thiamethoxam [31],
clothianidin [32], and thiacloprid [33]. Additionally, 2-Cl-pyridin-5-yl and 2-Cl-thiazol-5-yl moieties
play an important role in building the neonicotinoid insecticides; they are also the most insecticidal
moieties of neonicotinoid insecticides.
As shown in Figure 2, we aimed to introduce a neonicotinoid moiety into 1-position of
mesoionic pyrido[1,2-α]pyrimidinones and introduce a 2-Cl-pyridin-5-yl or 2-Cl-thiazol-5-yl moiety
Molecules 2018, 23, 1217; doi:10.3390/molecules23051217
www.mdpi.com/journal/molecules

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into 3-position of mesoionic pyrido[1,2-
α
]pyrimidinones to build some novel compounds. In this
paper, we reported the synthesis and their insecticidal activity against Aphis craccivora of two series
of mesoionic pyrido[1,2- ]pyrimidinone derivatives containing a neonicotinoid moiety. Moreover,
α
the label-free proteomics technique was used to study the protein differences after compound
I13 treatment.
Figure 1. The structure of Triflumezopyrim registered in China in 2016.
Figure 2. Design route of the target compounds.
2. Results and Discussion
2.1. 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,
1H-NMR, 13C-NMR, and HRMS. All copies of the spectrum for compounds I1
–I28 are available in
Supplementary Materials.

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Scheme 1. General synthetic procedure for the title compounds I1–I28.
2.2. 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
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 g/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 g/mL (Table 1). Meanwhile, at 500 g/mL,
compounds I1 I2 I6 I7 I8 I19 I20 I22 I23 I25, and I28 exhibited moderate activities (85%, 69%,
µg/mL,
µ
µ
µ
,
,
,
,
,
,
,
,
,
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 exhibit lower activity. Interestingly, when
R1 was benzyl with no substituents on the benzene ring, the corresponding compounds showed

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moderate insecticidal properties, such as I1 (mortality rate: 85%) and I2 (mortality rate: 69%). In a
word, the structures R1 and R2 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 1. Insecticidal activities of the title compounds I1–I28.
Compounds
Concentration (µg/mL)
Mortality Rate (%)
I1
I2
I3
I4
I5
I6
I7
I8
I9
I10
I11
I12
I13
500
500
500
500
500
500
500
500
500
500
500
500
500
200
100
50
500
500
500
500
500
500
500
500
500
500
500
500
500
500
500
500
200
100
50
85
69
31
23
0
51
77
51
28
46
35
43
100
100
92
30
16
0
23
49
0
58
62
39
62
58
0
62
27
46
62
100
100
100
100
100
100
100
I14
I15
I16
I17
I18
I19
I20
I21
I22
I23
I24
I25
I26
I27
I28
Imidacloprid
Triflumezopyrim
500
200
100
2.3. Label-Free proteomics Comparative Analysis
2.3.1. Analysis of Protein between Control and Treatment Groups
MaxQuant (version 1.5.2.8) search results identified 821 proteins, which were listed in
Supplementary Materials (Supplementary Materials 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).

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Figure 3. The up-regulated and down-regulated proteins in the control and treatment groups.
2.3.2. Bioinformatics Analysis
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).
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 [35–37]. 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.

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Figure 4. Some different expression proteins were grouped according to BP (A), CC (B), and MF (C).
3. Materials and Methods
3.1. 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.

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Intermediates
A
,
B, and
C
were prepared according to the reported methods [38
–
43]. First,
2-chloro-5-chloromethylpyridine was slowly added to the aqueous solution of NaHCO3 and
2-aminopyridine. Then, the mixture was refluxed for 5 h to afford intermediate . Meanwhile, a simple
A
formylation of 2-aminopyridine was created to give N-(pyridine-2-yl)formamide. Then, this compound
underwent a nucleophilic substitution reaction along with 2-chloro-5-(chloromethyl)thiazole to
produce intermediate B. Second, different substituted benzyl chlorides underwent at nucleophilic
substitution reaction with diethyl malonate. Then, efficient alkaline hydrolysis was conducted under
non-aqueous conditions by using dichloromethane/methanol (9:1) as solvent to provide 2-substituted
malonic acid, and the mixture of 2-substituted malonic acid, 2,4,6-trichlorophenol, and phosphorus
(V) oxychloride was refluxed for 3 h to obtain intermediate
C. To the solution of intermediate A or
B
in toluene (1 mmol, 25 mL), intermediate (1 mmol) was added and refluxed. The reaction was
C
monitored by TLC. After completion of the reaction, the solvent was removed under reduced pressure,
and the residue was purifed by column chromatography (ethyl acetate/methanol = 20/1) to give title
compound I.
3,4-dihydro-2,4-dioxo-1-((2-chloropyridin-5-yl)methyl)-3-benzyl-2H-pyrido[1,2-a]pyrimidin-1-ium-3-ide (I1).
Yellow solid; yield 44.7%; mp 65–67 ◦C. 1H-NMR (500 MHz, DMSO-d6)
δ 9.23 (d, 1H,
pyrido[1,2-a]pyrimidin Ar-H), 8.41 (s, 1H, 2-Cl-pyridin-5-yl 6-H), 8.20–8.19 (m, 1H, Ar-H), 7.73–7.70
(m, 2H, Ar-H), 7.47–7.44 (m, 2H, Ar-H), 7.31 (d, 2H, Ar-H), 7.20–7.18 (m, 2H, Ar-H), 7.17–7.08 (m, 1H,
Ar-H), 5.53 (s, 2H, Ar-CH2-N), 3.78 (s, 2H, Ar-CH2-C). 13C-NMR (125 MHz, DMSO-d6)
δ 159.6 (C=O),
154.3 (C=O), 149.7 (C=N, 2-Cl-pyridin-5-yl), 149.0 (Cl-C-N, 2-Cl-pyridin-5-yl), 146.3, 143.6, 142.3, 138.8,
131.9, 131.4, 128.7 (2C, Benzyl), 128.2 (2C, Benzyl), 125.7, 124.6, 116.8, 114.5, 92.8, 42.7 (CH2), 30.8 (CH2).
ESI-HRMS (m/z): calculating for C21H16ClN3O2 [M + H]+ 378.1004, we obtained 378.0996.
3.2. 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.
3.3. Proteomics
3.3.1. 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) f◦or 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].
3.3.2. 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

Molecules 2018, 23, 1217
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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].
3.3.3. 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
Column, 3
µ
L peptide samples. They were desalted on a ChromXP Trap column (Nano LC TRAP
m C18-CL, 120 A, 350 0.5 mm, Foster City, CA, USA). Then, the eluted samples
15 cm, Foster City,
µ
µm
×
were placed into column-Nano LC C18 reversed-phase column (3C18-CL, 75
µm
×
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 [55–57] 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.
4. 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.
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/23/5/1217/
s1, Table S1: identification of total protein, Table S2: identification of differentially expressed proteins, Table S3:
results of differential protein GO analysis.
Author Contributions: J.P. and D.H. conceived and designed the experiments. J.P., L.Y., and D.L. performed the
experiments. J.P. and D.L. analyzed the data. J.P. and D.H. wrote the paper.
Funding: This research was funded by National Natural Science Foundation of China (No. 21562013).

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Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds I1–I28 are available from the authors.
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