Synthesis and biological activity of 1,4-pentadien-3-one derivatives containing triazine scaffolds

Instruments and chemicals

Melting points were determined using an XT-4 digital melting-point apparatus (Beijing Tech. Instrument Co., Beijing, China) and readings were uncorrected. ¹H NMR , ¹³C NMR and ¹⁹F NMR spectra were recorded on a 400 MHz spectrometer (Swiss Bruker, Fällanden, Switzerland) with DMSO and CDCl₃ as the solvent and tetramethylsilane as the internal standard. The course of the reaction was monitored by thin-layer-chromatography analysis on silica gel GF₂₅₄ (Qingdao Haiyang Chemical Company, Ltd., Qingdao, China), and spots were visualized with ultraviolet (UV) light. High-resolution mass spectrometry (HRMS) was conducted by using a Thermo Scientific Q Exactive (Thermo Scientific, Missouri, USA). The molecular docking was performed by using DS-CDocker implemented in Discovery Studio (version 4.5). All reagents and solvents were purchased from Chinese Chemical Reagent Company and were of analytical grade reagents. The synthetic route to1,4-pentadien-3-one derivatives containing triazine moiety was shown in Fig. 5.

General procedure for the synthesis of intermediates

A synthetic route to 1,4-pentadien-3-one derivatives containing a triazine moiety was designed and shown in Fig. 5. According to previously reported methods (Chen et al., 2019; Tang et al., 2019; Gan et al., 2017), intermediates 1 and 2 could be obtained. Using benzyl, biacetyl and thio-semicarbazide as the initial materials in acetic acid and water was stirred at 100−110 °C for 6–8 h to obtain the intermediate 3 (Tang et al., 2019).

General procedure for the synthesis of target compounds 4a-4r

Reaction mixture was added to a solution of intermediate 2 (12 mmol), intermediate 3

(10 mmol) and K₂CO₃ (30 mmol) in dimethylformamide and stirred at room temperature for

6–8 h. After the reaction was completed (monitored by TLC), the mixture was extracted with ethyl acetate (30 mL × 3). The solvent was removed under reduced pressure. Residue was purified by silica-gel column chromatography using petroleum ether/ethyl acetate (3:1 v/v) to obtain target compounds 4a–4r. The ¹H NMR, ¹³C NMR, ¹⁹F NMR and HMRS spectra of the target compounds 4a–4r are also provided in the Supplemental Information.

Bioactivity assay

Antibacterial activity in vitro

The antibacterial activities of the title compounds against Xanthomonas axonopodispv. citri (Xac), Xanthomonas oryzaepv. oryzae (Xoo) and Ralstonia solanacearum (R.s) were evaluated at 100 µg/mL using a turbidimeter (Tang et al., 2019; Zhang et al., 2018). This test method is provided in the Supplemental Information.

Antiviral activities in vivo

Using N. tabacun L. leaves under the same age as that of test subjects, the curative, protective and inactivation activities against TMV (in vivo) at a concentration of 500 µg/mL were evaluated by the half-leaf blight spot method (Chen et al., 2019).This test method is provided in the Supplemental Information.

Molecular docking

The molecular docking was performed by using DS-CDocker implemented in Discovery Studio (version 4.5). This test method is provided in the Supplemental Information.

Antibacterial activities in vitro

The antibacterial activities of target compounds have been evaluated by the turbidimeter test (Zhang et al., 2018; Tang et al., 2019). Results in Table 1 indicated that some of synthesized compounds exhibited appreciable antibacterial activities against Xoo, R.s and Xac at the concentrates of 100 µg/mL. Among these derivatives, 4n and 4p exhibited excellent bactericidal effect against Xoo, with inhibition rates of 60.5% and 56.5%, respectively, which were superior to bismerthiazol (BT, 56.1%). In addition, as demonstrated in Table 1, the designed compounds displayed certain bactericidal effect toward R.s. Studies on the inhibition effect of title compounds suggested that 4a, 4b, 4j and 4k exhibited excellent inhibition effect against R.s with the inhibition rates of 58.2, 53.9, 53.5 and 61.9%, respectively, which were better than that of BT (52.1%). We also noticed that compounds 4k (91.8%) and 4l (95.4%) exposed better antibacterial activity toward Xac than that of BT (70.5%).

To further understand the antibacterial activity of the title compounds, the EC₅₀ values of some title compounds were calculated and summarized in Table 2. Notably, compounds 4a, 4b, 4j and 4k exhibited good inhibition effects against R.s, with half maximal effective concentration (EC₅₀) values of ranging from 0.43–4.76 µg/mL, which were better than that of BT (EC₅₀ = 49.5 µg/mL). Meanwhile, compounds 4j and 4k showed remarkable antibacterial activities against Xac with the EC₅₀ values of 55.53 and 129.1 µg/mL, which were better than that of BT (EC₅₀ = 153.7 µg/mL).

Antiviral activities against TMV in vivo

The antiviral activities of the title compounds 4a–4r against tobacco mosaic virus (TMV) were evaluated by the half leaf method (Chen et al., 2019) and the results were summarized in Table 3 and Fig. 6. It was found that some of the title compounds exhibited good antiviral activity against TMV in vivo. Compounds 4f, 4k and 4l showed remarkable curative activity against TMV, with values of 53.8, 66.3 and 59.9%, respectively. Which were better than that of ningnanmycin (NNM, 45.7%). Meanwhile, compound 4 h (61.4%) exhibited excellent protection activity, also superior to NNM (53.4%). Overall, most of the compounds indicated general inactivation activity against TMV at 500 µg/mL.

Based on the previous bioassays, the EC₅₀ values of some title compounds were tested and are listed in Table 4. Compound 4a exhibited excellent inactivation activity against TMV, with the EC₅₀ values of 12.5 µg/mL, which was better than that of NNM (EC₅₀ = 13.5 µg/mL). Moreover, compounds 4k and 4l exhibited the preferably curative activity against TMV, with EC₅₀ values of 11.5 and 12.1 µg/mL, respectively, which were superior to that of NNM (EC₅₀ = 82.2 µg/mL).

Molecular docking studies

Molecular docking studies (Figs. 7 and 8) for 4a with tobacco mosaic virus coat protein (TMV-CP) (PDB code:1EI7). Molecular docking results revealed that compound 4a was the most preferred compound based on the analysis followed by 4d and so on (Table 3). Compound 4a binding orientation clearly is described by Figs. 7 and 8, it forms one hydrogen bond with PHEA:12 with highest docking score (2.49 Å) among the designed molecules and the glide energy was also less compared to others showing few hydrophobic interactions with specific residues like as TYRA:139, VALA:75, LYSB:268 etc.

Structure–activity relationships of antibacterial activities

The antibacterial results in Tables 1 and 2 also indicated that the different groups on R had significant effects on the antibacterial activities of the title compounds. Obviously, the presence of a C₆H₄Cl group can effectively enhance the antibacterial activity against Xac. For example, the compounds 4k and 4l, which contain R = 4-Cl- C₆H₄ and R = 2-Cl- C₆H₄ groups respectively, exhibited EC₅₀ values of 55.53 and 129.1 µg/mL, which were better than that of BT (EC₅₀ = 153.7 µg/mL). Meanwhile, when R was substituted with thiophene-2-yl and 4-Cl- C₆H₄ groups, the corresponding compounds 4a, 4b, 4j and 4k exhibit remarkable antibacterial activities against R.s, with the EC₅₀ values of ranging from 0.43–4.76 µg/mL, which were better than that of BT (EC₅₀ = 49.5 µg/mL).

Structure–activity relationships of antiviral activities

The antiviral bioassay results indicated that the title compounds showed excellent antiviral activity against TMV. The preliminary SAR results were dropped based on the anti-TMV activity (as shown in Tables 3 and 4). The results indicated that when R was the 4-NO₂- C₆H₄ (4f), 4-Cl- C₆H₄ (4k) or 2-Cl- C₆H₄ (4l) group, the corresponding title compounds exhibited good curative activities. Furthermore, when the R was 4-OMe- C₆H₄ group, the protective activity of corresponding compound 4h was better than that of NNM (EC₅₀ = 82.2 µg/mL), with an EC₅₀ values of 32.1 µg/mL.