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


 
 Literatures revealed that 1,4-pentadien-3-one and triazine derivatives exhibited a wide variety of biological activities. In order to develop highly bioactive molecules, in this study, a series of novel 1,4-pentadien-3-one derivatives containing triazine moieties were synthesized and their antibacterial and antiviral activities were investigated.
 
 
 
 A series of novel 1,4-pentadien-3-one derivatives containing triazine moieties were synthesized and characterized in detail via 1H NMR, 13C NMR and HRMS spectra. The antibacterial activities against Xanthomonas axonopodispv. citri (Xac), Xanthomonas oryzaepv. oryzae (Xoo) and Ralstonia solanacearum (R.s) were evaluated at 100 and 50 µg/mL using a turbidimeter and N. tabacun L. leaves under the same age as that of test subjects. The curative, protective and inactivation activities against tobacco mosaic virus (TMV) at a concentration of 500 µg/mL were evaluated via the half-leaf blight spot method.
 
 
 
 The bioassay results showed that some of the target compounds exhibited fine antibacterial activities against Xac and R.s. Particularly, half maximal effective concentration (EC50) values of some target compounds against R.s are visibly better than that of the positive control bismerthiazol (BT). Notably, compound 4a showed excellent inactivation activity against TMV with a EC50 value of 12.5 µg/mL, which was superior to that ofningnanmycin (NNM,13.5 µg/mL). Besides, molecular docking studies for 4awith tobacco mosaic virus coat protein (TMV-CP) showed that the compound was embedded well in the pocket between the two subunits of TMV-CP. These findings indicate that 1,4-pentadien-3-one derivatives containing triazine moieties may be potential antiviral and antibacterial agents.



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 , 13 C NMR and 19 F NMR spectra were recorded on a 400 MHz spectrometer (Swiss Bruker, Fällanden, Switzerland) with DMSO and CDCl 3 as the solvent and tetramethylsilane as the internal standard.The course of the reaction was monitored by thin-layer-chromatography analysis on silica gel GF 254 (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 K 2 CO 3 (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 1 H NMR, 13 C NMR, 19 F NMR and HMRS spectra of the target compounds 4a-4r are also 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 EC 50 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

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 EC 50 values of some title compounds were tested and are listed in Table 4. Compound 4a exhibited excellent inactivation activity against TMV, with the EC 50 values of 12.5 µg/mL, which was better than that of NNM (EC 50 = 13.5 µg/mL).Moreover, compounds 4k and 4l exhibited the preferably curative activity against TMV, with EC 50 values of 11.5 and 12.1 µg/mL, respectively, which were superior to that of NNM (EC 50 = 82.2µg/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 (2.49Å) 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 C 6 H 4 Cl group can effectively enhance the antibacterial activity against Xac.

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-NO 2 -C 6 H 4 (4f), 4-Cl-C 6 H 4 (4k) or 2-Cl-C 6 H 4 (4l) group, the corresponding title compounds exhibited good curative activities.Furthermore, when the R was 4-OMe-C 6 H 4 group, the protective activity of corresponding compound 4h was better than that of NNM (EC 50 = 82.2µg/mL), with an EC 50 values of 32.1 µg/mL.

Figure 6
Figure 6 Tobacco leaf morphology effects of the NNM and 4k, 4h and 4a against TMV in vivo.(A) The curative activity of the ningnanmycin (NNM).The left side of the leaf (al) was smeared with compound NNM, and the right side of the leaf (ar) was not treated with compound NNM.(B) The protective activity of the ningnanmycin.The left side of the leaf (bl) was smeared with compound NNM, and the right side of the leaf (br) was not treated with compound NNM.(C) The inactivation activity of the ningnanmycin.The left side of the leaf (cl) indicated that smeared with compound NNM, and the right side of the leaf (cr) indicated that not treated with compound NNM.(D) The curative activity of the compound 4k.The left side of the leaf (dl) was smeared with compound 4k, and the right side of the leaf (dr) was not treated with compound 4k.(E) The protective activity of the compound 4h.The left side of the leaf (el) was smeared with compound 4h, and the right side of the leaf (er) was not treated with compound 4h.(F) The inactivation activity of the compound 4a.The left side of the leaf (fl) was smeared with compound 4a, and the right side of the leaf (fr) was not treated with compound 4a.Full-size DOI: 10.7717/peerjochem.3/fig-6

Table 1 Inhibition effect of the some title compounds against Xoo, R.s and Xac. a
Notes.aAverage of three replicates.b A commercial agricultural antibacterial agent Bismerthiazol was used for comparison of antibacterial activities.BT, Bismerthiazol.

Table 2 EC 50 values of some title compounds against Xoo, Xac and R.s. a Tested bacterias
Notes.aAverage of three replicates.bA commercial agricultural antibacterial agent Bismerthiazol was used for comparison of antibacterial activities.BT, Bismerthiazol.concentration (EC 50 ) values of ranging from 0.43-4.76µg/mL, which were better than that of BT (EC 50 = 49.5 µg/mL).Meanwhile, compounds 4j and 4k showed remarkable antibacterial activities against Xac with the EC 50 values of 55.53 and 129.1 µg/mL, which were better than that of BT (EC 50 = 153.7 µg/mL).

Table 3 Antiviral activities of the target compounds against TMV in vivo at 500 µg
/mL. a The antiviral activities of the title compounds 4a-4r against tobacco mosaic virus (TMV) at 500 µg/mL are shown.

Table 4 EC 50 values of the 4a, 4d, 4h,4k and 4l against TMV in vivo. a
The EC 50 values some of the title compounds against TMV in vivo are shown.A commercial agricultural antiviral agent ningnanmycin was used for comparison of antiviral activities.NNM, ningnanmycin Notes.aAverage of three replicates.b