Synthesis, Crystal Structure and Bioactivities of N -(5-(4-chlorobenzyl)-1,3,5-Triazinan-2-Ylidene)Nitramide

: The compound N -(5-(4-chlorobenzyl)-1,3,5-triazinan-2-ylidene)nitramide (C 10 H 12 ClN 5 O 2 , M = 269.70) was synthesized and structurally conﬁrmed by 1 H NMR, 13 C NMR, HRMS and single-crystal x-ray di ﬀ raction. The crystal belongs to the monoclinic system with space group P 2 1 / c . The title compound consisted of a benzene ring and a 1,3,5-triazine ring. All carbon atoms in the benzene ring were nearly coplanar with a dihedral (C6–C5–C10 and C7–C8–C9) angle of 1.71 ◦ and all non-hydrogen atoms of the 1,3,5-triazine ring were not planar, but exhibited a half-chair conformation. The crystal structure was stabilized by a strong intramolecular hydrogen bonding interaction N(3)–H(3) ··· O(2) and three intermolecular hydrogen bonding interactions, N(2)–H(2) ··· O(1), N(2)–H(2) ··· N(4) and N(3)–H(3) ··· Cl(1). The preliminary bioassay showed that the title compound showed not only aphicidal activity against Sitobion miscanthi (inhibition rate: 74.1%) and Schizaphis graminum (77.5%), but also antifungal activities against Pythium aphanidermatum (62.0%). These results provide valuable guidelines for the design and synthesis of novel aphid control agents and fungicides.

As part of our ongoing work in exploring triazine active chemical structures, we noticed that a type of 2-nitroimino-hexahydro-1,3,5-triazine (NHT) derivatives displayed biological activity against the apterous adult aphids of M. persicae [21,22] and Acyrthosiphon pisum [23]. In the view of these facts and in order to further search for NHT derivatives with high bioactivity and broad-spectrum, the titled compound N-(5-(4-chlorobenzyl)-1,3,5-triazinan-2-ylidene)nitramide (1), was designed by introducing benzyl group active substructure into NHT scaffold. The Compound 1 was synthesized through combining nitro guanidine, formaldehyde and 4-chlorobenzylamine via the Mannich reaction using the one-pot method in protic solvent (Scheme 1). The structure of the corresponding compound was characterized by 1 H NMR, 13 C NMR, HRMS and single-crystal x-ray diffraction. Furthermore, the insecticidal activity against different aphid species was evaluated. Moreover, compounds containing NHT group have been discovered to showed antifungal activity [24][25][26]. Herein, we have also estimated the antifungal activity of Compound 1. Scheme 1. Synthetic approach of the title Compound 1.

General Techniques
Melting point of the Compound 1 was determined on an X-5 binocular (Fukai Instrument Co., Beijing, China) with an uncorrected thermometer. 1 H NMR spectra were measured on a Bruker DPX300 spectrometer (Bruker, Bremen, Germany). Chemical shifts were reported in δ (ppm) with TMS as the internal standard and DMSO-d6 as the solvent. 13 C-NMR spectra were obtained by using a Bruker DPX300 spectrometer (75 MHz) with DMSO-d6 as a solvent. The chemical shifts (δ) were reported in parts per million using the solvent peak. High-resolution mass spectral data were acquired by a FTICR-MS Varian 7.0 T FTICR-MS instrument (Varian, Palo Alto, CA). A single-crystal x-ray structure was recorded on a Gemini E x-ray single crystal diffractometer (Rigaku, Tokyo, Japan). Nitro guanidine, formaldehyde and 4-chlorobenzylamine were purchased from Beijing Ouhe Technology Co., Ltd. (Beijing, China). All the other reagents were acquired from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China) and used without further purification.

Synthesis of N-(5-(4-Chlorobenzyl)-1,3,5-Triazinan-2-Ylidene)Nitramide
The target Compound 1 was prepared according to a modified procedure based on the published methods [27]. The synthetic approach of Compound 1 is shown in Scheme 1. To a solution of 4-chlorobenzylamine (58 mmol) and nitro guanidine (48 mmol) in ethanol (20 mL), 37% formaldehyde (120 mmol) was added dropwise. The reaction mixture was stirred at 60 • C for 6 h. After it was cooled to room temperature, the mixture was filtered and the filtrate was washed with cold ethanol and acetone, respectively, then dried under infrared lamp to obtain the white solid Compound 1 with a yield of 65.3%. m.p.: 201-203 • C. 1

Structure Determination
Single crystals suitable for x-ray diffraction were obtained from slow evaporation of a solution of the title compound 1 in dichloromethane/petroleum ester (v/v = 3/1) at temperature of 4 • C. Compound 1 exists in the form of colorless crystals. A crystal of Compound 1 (0.36 mm × 0.20 mm × 0.14 mm) was selected for data collection and mounted in inert oil, which was transferred to the cold gas stream of the Gemini E x-ray single crystal diffractometer (Rigaku, Tokyo, Japan) equipped with a graphite-monochromatic µMoKα radiation (λ = 0. 0.71073 Å) at temperature 109(10) K. In a range of 6.59 < 2θ < 58.972 • , a total of 9949 reflections were collected by using an ω scan mode, of which 3246 were unique with R int = 0.0332 and 2794 were observed with I > 2σ(I). The structure of Compound 1 was solved via Direct Methods and the solutions were refined by full-matrix least squares techniques on F 2 by SHELXL-2014 program [28]. All non-hydrogen atoms were refined anisotropically; the hydrogen atoms were located theoretically. The final R = 0.0468, wR = 0. parameters. Crystal data and structure refinement data of Compound 1 are shown in Table 1.

Aphicidal Activity
The in vivo aphicidal activities of Compound 1 against Myzus persicae, Sitobion miscanthi, Rhopalosiphum padi, Schizaphis graminum and Metopolophium dirhodum were measured using the reported method [29,30]. Compound 1 was dissolved in DMSO to a concentration of 2000 mg/L and then diluted to 200 mg/L with 0.05% Triton X-20. Wheat seedlings (for wheat aphids) or cabbage leaf discs (for M. persicae) were dipped into the test solution for 15 s. And then, the seedlings or discs were infested with 20 ± 3 apterous adult aphids and incubated under constant temperature (25 ± 1 • C) and light period (light : dark = 8:16) for 48 h. The number of dead aphids was then recorded, and the inhibition rates were corrected using Abbott's formula [31]. Each experiment was conducted in triplicates. The LC 50 values were also determined based on the preliminary aphid mortality rates. The commercial insecticide Pymetrozine was used as a positive control while the solvent was set as a negative control.

Antifungal Activity
The in vitro antifungal activities of the Compound 1 were evaluated against six plant fungal pathogens (Rhizoctonia solani, Pythium aphanidermatum, Valsa mali, Botrytis cirerea, Fusarium moniliforme, Alternaria solani). The mycelium growth rate method was used according to references [32,33]. Compound 1 was dissolved in DMSO to prepare the 10 mg/mL stock solution, then mixed with PDA (Potato Dextrose Agar) medium to a concentration of 50 mg/L and was poured into sterilized Petri dishes. After the dishes were cooled, the mycelia disks were inoculated in the center of the Petri dishes and incubated at 25 • C. Each experiment was repeated three times. After 2-3 d of culturing, the colony diameter of each strain was measured. The commercial fungicide, Difenoconazole, with broad spectrum against fungus was used as the positive controls.

Spectroscopic Properties
The structure of Compound 1 was confirmed by 1 H NMR, 13 C NMR and HRMS analysis. In the 1 H-NMR spectrum, one wide single peak with chemical shifts of δ 8.79 ppm exhibited the presence of N-H proton. The signals of the proton in the benzene ring were clearly discovered at δ 7.34-7.40 ppm. The protons of two methylene in the NHT ring and one methylene connected to the benzene ring were observed at 4.24 ppm and 3.78 ppm, respectively. The four methylene protons in 1,3,5-triazine ring had the same chemical shift. In the 13 C NMR spectrum, the carbons of C2/C3 in NHT ring, C6/C10 and C7/C9 in benzene ring appeared as doublets at 59.58 ppm, 130.69 ppm and 128.44 ppm, respectively. The CH 2 carbon C4 and the imino carbon C1 located the highest (53.25 ppm) and the lowest (155.80 ppm) field strength, respectively. The recorded HRMS spectral data of Compound 1 were in good accordance with the theoretical value. Symmetry transformations used to generate equivalent atoms: #1: -x + 2, -y, -z + 2; #2: -x + 2, -y, -z + 2; #3: x, -y + 1/2, z + 1/2.

Aphicidal Activity
The aphicidal activity of Compound 1 and the positive control Pymetrozine against M. persicae, S. mischanthi, R. padi, S. graminum and M. dirhodum are shown in Table 4. The preliminary bioassay results (at a concentration of 200 mg/L, for 48 h) indicated that Compound 1 exhibited insecticidal activity against all of the tested aphid species. The aphicidal activities against M. persicae, R. padi and M. dirhodum were moderate, with inhibition rates of 58.5%, 63.5% and 51.0%, respectively. Its inhibition rates of S. mischanthi and S. graminum reached 74.1% and 77.5%. However, the aphicidal activities of Compound 1 were lower than that of commercial Pymetrozine. The structure of Compound 1 showed a partially similar features to neonicotinoids (Figure 3: 1, 2, 3 and 4 represent aromatic heterocycle, flexible linkage, electron-withdrawing group and hydro-heterocycles or guanidine/amidine, respectively). It contained parts 3 and 4, but did not contain parts 1 and 2. However, the structure of control Pymetrozine was screened from many compounds. It is highly effective against aphids via blockage of stylet resulting in irreversible stop of feed [51]. The structure property of Compound 1 might lead to lower aphicidal activity than commercial Pymetrozine. In the future, introduction of aromatic heterocycle on part 1 and flexible linkage on part 2 to the scaffold structure of Compound 1 are recommended. On the basis of the primary experimental results, aphid species exhibiting a mortality rate higher than 70% were chosen to determine the LC 50 values. As shown in Table 5, Compound 1 exhibited a high aphicidal activity against S. miscanthi and S. graminum, with LC 50 values of 47.8 mg/L and 33.6 mg/L, respectively. However, the aphicidal activities of Compound 1 were lower than Pymetrozine with LC 50 values of 13.8 mg/L and 8.1 mg/L, respectively.    Table 6. The data suggested that all compounds had weak to moderate antifungal activity. The preliminary bioassay indicated that Compound 1 exhibits weak inhibition activity towards R. solani, V. mali and F. moniliforme. Its inhibition rates of P. aphanidermatum, B. cirerea and A. solani reached 62.0%, 56.4% and 56.1% at 50 mg/L, respectively. Unfortunately, Compound 1 showed activities sometime comparable but usually lower activities for these plant fungal pathogens compared with the Difenoconazole control. However, these results indicated that Compound 1 could be further used as a lead compound to develop novel fungicides, particularly against P. aphanidermatum. Thus starting from lead Compound 1, further studies could be envisaged and searched by intermediate derivatization approach, an effective method for the discovery of new biologically active molecules [52], by introduction of active substructure or by synthesis of new analogues and reporting the structure activity relationships.

Conclusions
In summary, the compound, N-(5-(4-chlorobenzyl)-1,3,5-triazinan-2-ylidene)nitramide, has been prepared by Mannich reaction and characterized by 1 H NMR, 13 C NMR, HRMS and single-crystal x-ray structural determination. The biological activity results showed that the title compound, Compound 1, had favorable insecticidal activity against the aphids of S. miscanthi and S. graminum and exhibited moderate antifungal activities. The bioassay results demonstrate that this compound has a wide range of biological activities. This study offered valuable clues and will lay the foundation towards the design and synthesis of novel aphid control agents and fungicides.