Design, synthesis, antiviral bioactivities and interaction mechanisms of penta-1,4-diene-3-one oxime ether derivatives containing a quinazolin-4(3H)-one scaffold

Background penta-1,4-diene-3-one oxime ether and quinazolin-4(3H)-one derivatives possess favorable agricultural activities. Aiming to discover novel molecules with highly-efficient agricultural activities, a series of penta-1,4-diene-3-one oxime ether derivatives containing a quinazolin-4(3H)-one scaffold were synthesized and evaluated for their antiviral activities. Result Antiviral bioassays indicated that some title compounds exhibited significant antiviral activity against tobacco mosaic virus (TMV). In particular, compounds 8c, 8j and 8k possessed appreciable curative activities against TMV in vivo, with half-maximal effective concentration (EC50) values of 138.5, 132.9 and 125.6 μg/mL, respectively, which are better than that of ningnanmycin (207.3 μg/mL). Furthermore, the microscale thermophoresis experiments (MST) on the interaction of compound 8k with TMV coat protein (TMV CP) showed 8k bound to TMV CP with a dissociation constant of 0.97 mmol/L. Docking studies provided further insights into the interaction of 8k with the Arg90 of TMV CP. Conclusions Sixteen penta-1,4-diene-3-one oxime ether derivatives containing a quinazolin-4(3H)-one scaffold were designed, synthesized, and their antiviral activities against TMV were evaluated. Antiviral bioassays indicated that some target compounds exhibited remarkable antiviral activities against TMV. Furthermore, through the MST and docking studies, we can speculate that 8k inhibited the virulence of TMV by binding Arg90 in TMV CP. These results indicated that this kind of penta-1,4-diene-3-one oxime ether derivatives containing a quinazolin-4(3H)-one scaffold could be further studied as potential alternative templates in the search for novel antiviral agents. Electronic supplementary material The online version of this article (10.1186/s13065-019-0547-1) contains supplementary material, which is available to authorized users.


Background
Tobacco mosaic virus (TMV) is an important plant virus that caused serious crop loss [1], and unfortunately, there are few effective antiviral agents to control the infection of TMV [2]. Ningnanmycin, exhibited a dual targeting ability against the TMV coat protein [3], is the best anti-TMV agent that was isolated from Streptomyces noursei var. [4]. However, its application in field trail is largely limited by its photosensitivity, water stickiness and high control costs [5,6]. Therefore, developing novel, highlyefficient, and environmentally benign antiviral agents remains a daunting task in pesticide sciences.

Chemistry
The infrared spectrum (IR), 1 H nuclear magnetic resonance ( 1 H NMR), 13 C nuclear magnetic resonance ( 13 C NMR) and high resolution mass spectrum (HRMS) spectra of title compounds are shown in Additional file 1. IR spectra exhibit characteristic absorptions at 1650-1500 cm −1 , which indicate the presence of -C=N-fragment. The signals at 1750-1650 and 1260-1210 cm −1 are attributed to the stretching frequencies of -C=O and -C-O-N=groups, respectively. In 1 H NMR spectra, multiplet signals at δ 8.20-6.50 ppm show the presence of protons in olefinic bonds and aromatic nucleuses, and a singlet at δ 5.50-5.00 ppm reveals the presence of -CH 2groups. Absorption signals at δ 155-165 and 65-75 ppm in 13 C NMR spectra confirm the presences of -C=Nand -CH 2 -groups, respectively. Noteworthily, the 1 H NMR and 13 C NMR spectra of title compounds also reveal that title compounds don't have chemical isomers, which are consist with a result described in our previous work [22]. In the HRMS spectra of target compounds show characteristic absorption signals of [M+H] + ions that is consistent with their molecular weight.

Antiviral activity screening of title compounds against TMV in vivo
Using N. tabacun L. leaves under the same age as the test subjects, the curative and protective activities against TMV in vivo at 500 μg/mL were evaluated by the halfleaf blight spot method [14,18,34,35] and the obtained results were listed in Table 1. Preliminary bioassays indicated that the curative and protective effects of title compounds against TMV at 500 μg/mL ranged 31.7-59.1% and 39.4-69.8%, respectively. Among them, the curative effects of compounds 8c, 8d, 8e, 8j and 8k against TMV were 55.9, 53.4, 54.6, 55.4 and 59.1%, respectively, which are better than that of ningnanmycin (51.8%). In addition, compounds 8c, 8j and 8k exhibited significant protection effects against TMV at 500 μg/mL, with corresponding inhibitory rates of 69.8, 67.7 and 72.0%, respectively, which are superior to that of ningnanmycin (65.7%).
Based on the preliminary bioassays in Table 1, the curative activities of compounds 8c, 8j and 8k against TMV at 500, 250, 125, 62.5 and 31.25 μg/mL were respectively tested to obtain the corresponding EC 50 values. As showed in Table 2, compounds 8c, 8j and 8k showed excellent curative effects against TMV, with corresponding EC 50 values of 138.5, 132.9 and 125.6 μg/mL, respectively, which are better than that of ningnanmycin (207.3 μg/mL). These results indicated that this kind of penta-1,4-diene-3-one oxime ether derivatives containing a quinazolin-4(3H)-one scaffold could be further studied as potential alternative templates in the search for novel antiviral agents.

The expression and purification of TMV CP
pET28a-TMV CP vector were constructed and preserved in our lab [36,37]. TMV CP genes were overexpressed in pET28a-TMV CP vector when the final concentration was increased to 0.8 mM isopropoyl-βd-galactopyranoside (IPTG) and the solution was left overnight at 16 °C. More than 90% of the TMV CP protein was eluted. Then, the dealt proteins were loaded in desalting column, in a buffer containing 10 mM PB and 100 mM sodium chloride pH 7.2, the retention time of the TMV CP protein was determined with 19.5 kDa with His-tags.

The binding studies of TMV CP and 8k
We studied the interactions between 8k and TMV CP using Monolith NT. 115 (Nano Temper Technologies, Germany) [38,39]. The results showed that the 8k bound to TMV CP with a dissociation constant (Kd) of 0.97 ± 0.69 µM. An affinity between 8k and TMV CP was far greater than the affinity between ningnanmycin (Control) and TMV CP. Base on anti-TMV activities and MST results. We speculate TMV CP protein was a potential target of 8k. The affinity curves are shown in Fig. 2.

Molecular docking of 8k and TMV CP
To identify the 8k recognition sites in TMV CP, we performed molecular docking using the Gold method with 200 cycles. After optimization, we found that Arg90 in the TMV CP shared one hydrogen bonds with 8k, C=O-H-N = 3.24 Å (Fig. 3c). In our previously reported, Arg90 was an important residue for TMV RNA binding [38,39]. These analyses indicated that Arg90 was the key residue to infect the activity of 8k.

Chemistry
Melting points of synthesized compounds were determined under an XT-4 binocular microscope (Beijing Tech Instrument Co., China) and were not corrected. The IR were recorded from KBr disks using a Bruker VECTOR 22 spectrometer (Bruker, USA). NMR spectra were obtained on a JEOL-ECX500 NMR spectrometer (JEOL, Japan) at room temperature using tetramethylsilane as an internal standard. Reaction was monitored by thin-layer chromatography (TLC) on silica gel GF 245 (400 mesh). Mass spectral studies were performed on a quadrupole/electrostatic field orbitrap mass spectrometer (Thermo Scientific, USA). The micro thermophoresis of the compound and TMV CP was determined by a micro thermophoresis instrument (NanoTemper Tchnologies GmbH, Germany); the fluorescence spectroscopy of the compound interacting with TMV CP was determined by FluoroMax-4 fluorescence spectrometer (HORIBA Scientific, France). All reagents and reactants were purchased from commercial suppliers and were analytical grade or chemically pure. The synthetic route to penta-1,4-diene-3-one oxime ether derivatives containing a quinazolin-4(3H)-one scaffold was shown in Scheme 1. Intermediates 2 and 7 were prepared according to the reported methods [16,31].

Expression and purification of TMV CP
According to previous procedure described in the literature [36], The pET28a-TMV CP as a expression vector, which was stored at − 80 °C in our lab containing the full-length TMV CP gene. After overnight culture, the Escherichia coli strain BL21(DE3) containing the plasmid pET28a-TMV CP freshly transformed, and was transferred to 1 L Luria broth. At 37 °C in Luria-Bertani medium, the cells were grown which were supplemented with 50 μg/mL kanamycin and shaken at 200 rpm until the OD 600 was 0.8. At 16 °C, using 0.8 mM IPTG inducing the protein expression overnight, then centrifugation to obtained the cells, and stored at − 80 °C. When analyzed, the cells were resuspended in lysis buffer (500 mM NaCl, 20 mM PB, 5 mM β-mercaptoethanol, 30 mM imidazole and 5% glycerol, pH 7.2) and then use sonication to lysed at 4 °C. By centrifugation at 12,000g for 30 min at 4 °C, the lysate was clarified, the soluble supernatants were loaded onto a 5 mL Ni-NTA column (GE Healthcare, USA), and using a linear gradient of 30-350 mM imidazole (pH 7.2) to eluting the protein. using a desalting column (GE Healthcare, USA) attached to an AKTA purifier protein liquid chromatography system (GE Healthcare, USA), the crude protein was performed at 4 °C, and the fractions containing target protein with His-tags were pooled, we use ultrafiltration (10 kDa cut-off ) concentrated to a suitable concentration. Using a Genequant100 (GE Healthcare, USA) to determined the dealt protein concentration, stored at − 80 °C until further analysis.

Interaction studies between 8k and TMV CP
According to previously classic method, the binding was calculated for MST Monolith NT. 115 (Nano Temper Technologies, Germany) [38,39]. Using a NT-647 dye (Nano Temper Technologies, Germany) incubating 0.5 µM of purified recombinant proteins with A range of ligands from 0 to 5 µM were for 5 min, and using the final concentration of 20 nM to the thermophoresis experiment.

General synthesis procedures for title compounds 8
A solution of intermediates 4 (1.50 mmol), intermediates 7 (1.80 mmol) and K 2 CO 3 (3.00 mmol) in acetonitrile (30 mL) was stirred under reflux for 4-5 h. Then, the mixture was filtered, the solvent was removed under vacuum, and the crude product was then separated by column chromatography with petroleum ether/ethyl acetate (V:V = 1:1) to obtain the title compounds 8a-8p.