Synthesis and antiviral bioactivities of 2-aryl- or 2-methyl-3-(substituted-Benzalamino)-4(3H)-quinazolinone derivatives

A simple and general method has been developed for the synthesis of various4(3H)-quinazolinone derivatives by the treatment of the appropriate 3-amino-2-aryl-4(3H)-quinazolinone with a substituted benzaldehyde in ethanol. The structures of the compoundswere characterized by elemental analysis, IR, (1)H-NMR and (13)C-NMR spectra. The title 2-aryl- or 2-methyl-3-(substituted-benzalamino)-4(3H)-quinazolinone compounds III-1~III-31 were found to possess moderate to good antiviral activity. Semi-quantitative PCR andReal Time PCR assays were used to ascertain the target of action of compound III-31against TMV. The studies suggest that III-31 possesses antiviral activity due to inductionof up-regulation of PR-1a and PR-5, thereby inhibiting virus proliferation and movementby enhancement of the activity of some defensive enzyme.

For this report we have designed and synthesized a series of 2-aryl-or 2-methyl-3-(substitutedbenzalamino)-4(3H)-quinazolinone derivatives III-1~III-31 and investigated their antiviral bioactivities.The synthetic route used is shown in Scheme 1. Substitution patterns and optimized yields are given in Table 3.The structures of these compounds were firmly established by well defined IR, 1 H-NMR, 13 C-NMR data and elemental analysis.Preliminary bioassay tests showed that some compounds displayed in vivo antiviral activity against TMV at 500 μg/mL.Scheme 1. Synthetic route to the title compounds.

Results and Discussion
Different homologues of 3-amino-2-aryl-4(3H)-quinazolinone II were prepared following a literature procedure [20].Reaction conditions were non-homogeneous and the use of an increased amount of hydrazine hydrate did not afford successful results.The reaction conditions for the synthesis of II-1 (R=H, R 1 =H, R 2 =Ph) were optimized in various solvents at different temperatures for different times and the results are shown in Table 1.An 87 % yield could be obtained when the reaction mixture was heated in pyridine at 116 °C for 0.5 h.In order to optimize the reaction conditions for the title compounds, the synthesis of III-1 (R=H, R 1 =3, 5-di-chloro，R 2 =Ph) was examined under different conditions.First, the role of the catalyst (HOAc) in accelerating the reaction rate was ascertained.While in the presence of catalyst, a 72% yield of III-1 was achieved in only 10 h (Table 2, entry 1), a yield of only 52% was obtained with a prolonged reaction period of 30 h in the absence of the catalyst (Table 2, entry 2).In addition, we also examined the effects of reaction time.When the reaction time was prolonged further to 20 h, no improvement (75 %) was obtained (Table 2, entry 3), as compared to that of 10 h (72 %).Also, it could be observed that the yield was significantly lower at room temperature (Table 2, entry 4).For different substituted benzaldehydes, under optimal conditions, as depicted in Table 3, III-1-III-31 could be obtained in 53-89% yields at 78 °C in ethanol in presence of HOAc as the catalyst.Representative analysis of spectral data: IR and 1 H-NMR of compound III-3.
The IR spectrum of III-3 (molecular formula C 22 H 14 F 3 N 3 O, m.w.393.37, structure and carbon numbering in Figure 1) over the 3060-3032 cm -1 range showed multiple weak absorption peaks corresponding to Qu-H and Ar-H stretching vibration absorption peaks.The strong absorption at 1671 cm -1 is due to the C=O stretching vibration and the moderate intensity absorption at 1607 cm -1 corresponds to a C=N stretching vibration.The 1591~1474 cm -1 absorptions are due to the skeleton vibration of the aryl and heterocyclic rings.The series of bands in the 1319, 1174, 1125 cm -1 region correspond to the para C-F stretching of the imine-Ar-CF group, the one at 843 cm -1 is due to a methylene linked to the benzene ring at the substituted 4-position.The absorption peaks at 766 and 696 cm -1 arise due to the phenyl-substituted at the 2-position in the quinazolinone.4.

Antifungal activity
The antiviral bioassay results are given in Table 5.It could be seen that these newly synthesized derivatives exhibit weak to good antiviral activities.At 500 μg/mL, the title compounds exhibited weak activities against Fusarium oxysporum, Valsa mali and Gibberella Zeae, which are lower than that of a hymexazol standard.

Antiviral activity
The in vivo bioassay results against TMV are given in Table 6.Ningnanmycin was used as reference antiviral agent.It was found that these compounds exhibit certain activities against TMV in vivo.The compounds III-31, III-16, III-3, III-1 and III-8 have relatively higher curative effect than the other compounds III-2, III-4, III-5, III-6, III-7, III-9-III-15, III-17 and III-30.The data listed in Table 6 indicate that antiviral activity depends on the nature of the substituents present in the title compound.When R was 4-CF 3 , the title compounds III-31 and III-16 showed curative rates of 55 and 54%, which was slightly higher than that of reference (54%) against TMV at 500 µg/mL.The other compounds displayed a slightly lower antiviral activity than that of the reference.

Product of PCR in PR-1a and PR-5 and its sequence identification
By following RT-PCR, the PCR product produced in 35 cycles was resolved on a 1.5% agarose gel.The purified products of PCR were sequenced by model ABI 310 DNA sequencer and were identified as the target gene by BLAST searching in Genbank (Figure 3).

Gene expression analysis of PR-1a and PR-5 in III-31-treated tobacco leaf
In vitro synthesized single-stranded cDNA from RNA samples were isolated from leaves of watertreated tobacco and the TMV-treated tobacco and the III-31-and TMV-treated tobacco.The differential expression analysis of the PR-1a and PR-5 gene was determined by semi-quantitative PCR and the relative quantification real-time PCR analysis.The mRNAs of PR-1a and PR-5 gene accumulated to detectable levels in III-31-and TMV-treated tobacco leaf, while no-detectable levels were reached in water-treated tobacco and the TMV-treated tobacco.The mRNA content of III-31-treated tobacco leaf for PR-1a gene started to increase after five days and reached a peak at the end of the 5 th day before falling to the normal level.In contrast, in TMV-treated tobacco leaf, no significant increase in the levels of gene expression was noticed (Figures 4-A, 5-A).The expression levels of PR-5 gene in III-31-treated tobacco rapidly increased and reached a peak within 5 th day after the inoculation and then started to decrease gradually.As depicted in Figure 4-B and Figure 5-B, III-31 treated tobacco leaves showed significant enhancement in the levels of gene expression as compared to TMV-treated tobacco leaves within 5 th day after the inoculation.In contrast, in TMV-treated tobacco leaf, no significant increase in the levels of gene expression was observed.

Conclusions
In summary, the presented new method of preparation of 4(3H)-quinazolinone Schiff base derivatives from appropriate 3-amino-2-aryl-4(3H)-quinazolinones and substituted benzaldehydes in ethanol is convenient, rapid and gives moderate yields.It was also found that the title compounds III-31, III-1, III-3, III-8, III-16 displayed good antiviral activity.Semi-quantitative PCR and real time PCR assay were evaluated to ascertain the target of action of compound III-31 against TMV.The studies suggest that III-31 possesses antiviral activity by induction up-regulation of PR-1a, PR-5 thereby inhibiting proliferation and movement of virus, by enhancing activity of some defensive enzyme.

General
The melting points of the products were determined on a XT-4 binocular microscope (Beijing Tech Instrument Co., China) and were not corrected.The IR spectra were recorded on a Bruker VECTOR 22 spectrometer on KBr disks. 1 H-and 13 C-NMR experiments (solvent DMSO-d 6 ) were recorded at 500 and 125 MHz, respectively, on a JEOL-ECX 500 NMR spectrometer at room temperature using TMS as an internal standard.Elemental analysis was performed on an Elementar Vario-III CHN analyzer.All reagents employed were of analytical reagent grade or chemically pure.All solvents were dried, deoxygenated and redistilled before use.

Preparation of Schiff base derivatives III-1~III-31
To a solution of the appropriate substituted benzaldehyde (1.0 mmol) in ethanol (15 mL), were added the 3-amino-2-aryl-4(3H)-quinazolinone (1.0 mmol) and a few drops of acetic acid (0.05 mmol).The reaction mixture was refluxed for 3-24h and the course of the reaction was monitored by TLC [petroleum ether/ethyl acetate (V/V=1:2)] to its completion.The reaction mixture was cooled.The crude product was recrystallized from 95% ethanol to give title compounds III-1~III-31.

Bioassays: Antifungal Bioassays
The antifungal activity of all synthesized compounds III-1-III-31 was tested against three pathogenic fungi, namely Fusarium oxysporum, Gibberella zeae, and Valsa mali, by the poison plate technique [22].Compounds III-1-III-31 were dissolved in acetone (10 mL) before mixing with Potato Dextrose Agar (PDA, 90 mL).The final concentration of compounds III-1-III-31 in the medium was fixed at 500 μg/mL.Three kinds of fungi were incubated in PDA at 25±1 °C for 5 days to get new mycelium for antifungal assay, then a mycelia disk of approximately 0.45 cm diameter cut from the culture medium was picked up with a sterilized inoculation needle and inoculated in the center of PDA plate.The inoculated plates were incubated at 25±1°C for 5 days.Acetone in sterilized distilled water served as control, while hymexazole was used as positive control For each treatment, three replicates were carried out.The radial growth of the fungal colonies was measured on the sixth day and the data were statistically analyzed.The in vitro inhibiting effects of the test compounds on the fungi were calculated by the formula CV = A B A − , where A represents the diameter of fungi growth on untreated PDA, B represents the diameter of fungi on treated PDA, and CV represents the rate of inhibition.

Antiviral Bioassays
The leaves of Nicotiana tabacum.L growing at the same ages were selected.The tobacco mosaic virus with the concentration of 6×10 -3 mg/mL was dipped and inoculated on the whole leaves.Then the leaves were washed with water and dried.The compound solution was smeared on the left side and the solvent was smeared on the right side for control.The local lesion numbers were then recorded 3-4 days after inoculation [23].For each compound, three times of repetition were conducted to ensure the reliability of the results.

Isolation of total RNA from tobacco leaf
Trizol kit was used according to the standard protocol for total RNA isolation.Prior to RT-PCR, the total RNA samples were treated with DNase I for 10 min and quantified by spectrophotometry and identified by agarose gel electrophoresis [24].

RT-PCR assay
cDNA was synthesized with oligo (dT) 18 complementary at the 3' end of mRNA as a primer.Total RNA (1μg) was used for temple of first-strand cDNA synthesis using extend reverse transcriptase.Reverse transcription was carried out at 37 o C for 1 h.The single-stranded DNA mixture was used as template in PCR.The primers for PCR amplification are shown in Figure 3.The PCR were performed in Tris-HCl buffer (10 mM, pH 8.3), KCl (50 mM), MgCl 2 (1.8-2.0 μl), dNTPs (0.02 μM), primers (0.04 μM), DNA polymerase (1 U).PCR amplification steps consisted of a preliminary denaturation step at 94 o C for 1 min, followed by 35 cycles at 94 o C for 40 seconds, at 58 o C for 40 seconds and at 72 o C for 50 seconds on icycle of BioRad.PCR products were separated on 1.5% agarose gel in 0.5×TBE buffer and visualized under UV light after staining with ethidium bromide [25]

Products of PCR sequencing
The purified PCR products were sequenced on an automated DNA sequencer by model ABI 310 DNA sequencer.The sequencer analysis was carried out using the chromas223 software and BLAST network services at the National Center for Biotechnology information (NCBI) Genbank.

Semi-quantitative PCR for gene expression
In order to assess relative expression levels of target-gene in water-treated tobacco and (R)-4p-and TMV-treated tobacco and tobacco by inoculated TMV only, semi-quantity PCR consisting of 20 cycles (within the logarithmic range of amplification of gene) with putting primer of β-actin serving as an internal reference gene was employed for the study.The amplified products were analyzed on a 1.5% agarose gel by the method of F. Mohamed [26].

The relative quantification real-time PCR for expression of the target gene
The relative quantification real-time PCR was carried out with iCycler IQ according to manufacturer's protocol with primer of β-actin serving as an internal reference gene.Precautions were taken to ascertain reliable quantitative results: Log-linear dilution curves were performed with primers for the target gene as well as with primers for the β-actin.Reactions performed without reverse transcriptase or without template did not result in any product.Reactions were conducted in 50 μL final volumes with IQ Supermix (25 μL) containing SYBR Green and cDNA as template (2μg), and primer mixture (40 pM) for each gene.PCR cycles were as follows: 2 min at 94 C T being the cycle threshold.Rates of stimulation of RNA expression were calculated from the ΔC T values at various time points [27].

Figure 1 . 3
Figure 1.The structure and carbon atom numbering of compound III-3

8
13 C-NMR of compound III-The chemical structure of compound III-8 and the assignment of carbon atoms are shown in Figure 2.This compound (molecular formula C 22 H 14 F 3 N 3 O, m.w.393.37) contains 22 carbon atoms.Among them, there are two equivalent pairs, C-17, C-21 and C-18,C-20.The 13 C chemical shift values of the 20 non equivalent carbons are assigned and shown in Table

Figure 2 .
Figure 2. The structure and carbon atoms assignment of compound III-8.

Figure 4 .
Figure 4.The real time PCR effects of PR-1a gene.
, MMLV reverse transcriptase and RT-PCR kit were purchased from TAKARA Biotechnology (Dalian) CO., LTD.DNA Marker pUC/Msp I was purchased from MBI Company, Ltd.Primer was designed with Beacon Designer software.Oligo (dT) 18 and primers were synthesized by Shanghai Sangon Biotechnology Company, Ltd.
o C, followed by 40 cycles each of 40 seconds duration at 94 o C, 40 seconds at 58 o C, and 50 seconds at 72 o C. By following PCR, 110 steps for melt curve analysis were completed in 10 seconds at temperature ranging from 40 o C to 95 o C. The amplification efficiency was 95-99% for PR-1a and PR-5 gene, respectively (The figure of standard curve are not shown).Each target gene peak was assigned an arbitrary quantitative value correlated to the β-actin gene peak, according to the formula: ΔΔC T =ΔC T (test) -ΔC T (calibrator)

Table 1 .
The effect of reaction conditions on yield of intermediate II-1 (R=H, R 1 =H, R 2 =Ph).

Table 2 .
The effect of reaction conditions on yield of title compound III-1.

Table 3 .
Substituents and yields of title compounds III-1-III-31 under optimized conditions.

Table 6 .
The curative effects of the new compounds III against TMV in vivo.