Full Paper Synthesis and Antiviral Bioactivities of α-Aminophosphonates Containing

A simple, efficient, and general method has been developed for the synthesis of various alpha-aminophosphonate derivatives 4a-4l by treatment of substituted benzaldehydes and aniline with bis(2-methoxyethyl)- or bis(2-ethoxyethyl) phosphite under microwave irradiation without solvents and catalysts. The products were characterized by elemental analysis, IR, 1H-NMR, 13C- and 31P-NMR spectra. The X-ray crystallographic data of the representative compound 4l was determined. The new alpha-aminophosphonate derivatives were found to possess moderate to good antiviral activity.


Introduction
α-Aminophosphonate derivatives have attracted much attention in medicinal and pesticide chemistry over the past two decades due to their biological activities.Some of them have been used as potent enzyme inhibitors [1], antimicrobial [2], antitumor [3] and antiviral agents [4].In our search for new potential plant antiviral agents, we have reported the design and synthesis of a series of α-aminophosphonate compounds, among which some were found to possess moderate to good bioactivity [5,6].Recently, attention has been focused on their potential as antiviral agents after the discovery of the activity of compounds containing the methoxyethyl moiety [7].We now report the design and synthesis of a series of novel α-aminophosphonate compounds containing alkoxyethyl moieties and the investigation of their bioactivity.The synthetic route is shown in Scheme 1.The structures of compounds 4a-4l were firmly established by IR, 1 H-, 13 C-and 31 P-NMR and elemental analysis.The X-ray crystallographic data of compound 4l is also provided.Preliminary bioassay tests showed that some of these compounds displayed in vivo antiviral activity against Tobacco Mosaic Virus (TMV) at 500 µg/mL.Scheme 1 Synthetic route to the title compounds.

Results and Discussion
In order to optimize the reaction conditions, the synthesis of 4a was carried out under a variety of conditions.First, it was determined that microwave irradiation should be used, as the reactions without microwave activation were much slower and, for example, the product was obtained in a yield of only 64.9 %, after 4h of conventional heating as compared with the yield of 83.5% obtained after 10 min.under microwave irradiation (Table 1, entries 2, 11).In addition, we also examined the effects of reaction time on the reactions.When the reaction times were increased from 5 min.to 10 min., 15 min.and 20 min., 4a was obtained in 78.9, 83.5, 82.7 and 81.8 % yield, respectively (Table 1, entries 1-4).When the reaction time was prolonged further to 25 min.(entry 5), no additional improvement was noted (82.8 % yield) as compared to that obtained after 10 min.(83.5 % yield, entry 2).The effect of reaction temperature was also studied.It could be seen that the yields were relatively lower when the reactions were carried out at 60 or 80 °C (Table 1, entries 6, 7) than at 100 °C (entry 2).A lower yield was also observed when the reaction system was heated to 120 °C, and no yield was observed when the reaction system was heated to 140 °C (Table 1, entries 9, 10).Consequently, it is better for the reaction to be carried out at 100 °C than at lower or higher temperatures.Using the optimized conditions, the best results were obtained when bis(2-methoxyethyl) phosphite or bis(2-ethoxyethyl) phosphite were treated with 1 equiv. of substituted benzaldehyde and 1 equiv. of substituted aniline under microwave conditions without solvent or catalyst at 100 °C for 10 min.Under these conditions, the Mannich reactions proceeded smoothly, and the results are summarized in Table 2.The structures of title compounds 4a-4l were established on the basis of their spectroscopic data.They showed IR absorption bands at 3200-3500 (NH) and 1520-1616 cm -1 (C=C) (aromatic ring skeletal vibration), while the absorption at 1220~1250 cm -1 was assigned to the P=O stretching absorption bands and that at 990~1100 cm -1 to the C-O stretching absorption bands in the P-O-C group.In the 1 H-NMR all phenyl protons showed multiplets at 6.19-7.78ppm.The chemical shifts of the ester PCH were about 4.85-6.17ppm, respectively.

Antifungal activity bioassay
The antifungal bioassay results are given in Table 3.It can be seen that the newly synthesized alkoxyethyl α-aminophosphonate derivatives 4a-4l exhibit weak antifungal activities at 500 µg/mL towards Fusarium oxysporum, Valsa mali and Gibberella zeae, which were obviously lower than that of a hymexazol standard.

Antiviral activity bioassay
The results of the in vivo bioassay against Tobacco Mosaic Virus (TMV) are given in Table 4. Ningnanmycin was used as the reference antiviral agent.The results indicated that the antiviral activity depended on the substituents present.When R 1 is H, R 2 is a 4-trifluoromethyl group and R 3 is CH 3 , (4b) the compound showed a 56.5 % curative rate against TMV at 500µg/mL, slightly higher than that of reference (53.8 %).The inhibitory rates of compounds 4i, 4d, 4e, 4c and 4g at the same 500µg/mL concentration were 53.1 %, 51.6 %, 51.3 %, 49.2 % and 46.0%, respectively.These compounds all have an antiviral activity slightly lower than that of the reference compound.The other compounds 4a, 4k, 4f, 4h, 4j and 4l exhibited a weak anti-TMV bioactivity, with inhibitory rates of 38.8%, 34.3 %, 34.2%, 25.0 %, 16.2 % and 13.8 %, respectively, at 500µg/mL.

Crystal Structure Analysis
As it can be seen from the X-ray single crystal analysis of 4l (Figure 1), the dihedral angle between the C(8)-C(7)-N(2)-C(5) plane is -72.3(2)°.The N-H … O type of intermolecular interactions plays a major role in stabilizing the molecules in the unit cell.As shown in the packing diagram of this compound (Figure 2), there is a N ).In the solid state, the above hydrogen bonds connecting the molecules form hydrogen bond networks which stabilize the crystal structure.

Conclusions
A new method is presented for the formation under microwave irradiation conditions of α-aminophosphonates containing alkoxyethyl moieties.The method offers several advantages: an easy, rapid, one-pot reaction, environmental-friendliness and good yields.It was also found that the title compounds 4b, 4i, 4d and 4e displayed good antiviral activity.

General
The melting points of the products were determined on a XT-4 binocular microscope (Beijing Tech Instrument Co., P.R. China) and are uncorrected.IR spectra (KBr disks) were recorded on a Bruker Vector 22 spectrometer. 1H-and 13 C-NMR spectra (solvent CD 3 COCD 3 ) were recorded at room temperature on a JEOL-ECX 500 NMR spectrometer operating at 500 and 125 MHz, respectively, using TMS as internal standard. 31P-NMR spectra were measured with 85% H 3 PO 4 as external reference. .The reference sample was prepared by sealing a capillary containing 85% H 3 PO 4 in a 5 mm NMR tube inside which a suitable amount of CDCl 3 was added for field locking.Elemental analysis was performed on an Elementar Vario-III CHN analyzer.Microwave reactions were performed on a Discovery TM LabMate Focused Microwave Synthesizer (50 W power). Analytical TLC was performed on silica gel GF254.All reagents were analytical reagent grade or chemically pure.Solvents were dried, deoxygenated and redistilled before use.Dialkyoxyethyl phosphites were synthesized according to the literature method [8].

Preparation of Alkyloxyethyl-containing α-Aminophosphonates 4a-4l
Substituted benzaldehyde (5 mmol) and aniline (5 mmol) and di(2-methoxyethyl) phosphite or di(2-ethoxyethyl) phosphite were placed in a microwave tube, which was the sealed and placed in the Discovery TM synthesizer and irradiated at 100 °C and 50 W for 10 min.Completion of the reaction was checked by TLC.The reaction mixture was cooled and the crude product was recrystallized from 95% ethanol to give title compounds 4a-4l.(2-methoxyethyl)

Bioassays: Antifungal Bioassays
The antifungal activity of all synthesized compounds 4a-l was tested against three pathogenic fungi, namely Fusarium oxysporum, Gibberella zeae, and Valsa mali, by the poison plate technique [9].Test compounds were dissolved in acetone (10 mL) before mixing with Potato Dextrose Agar (PDA, 90 mL).The final concentration of compounds 4a-l 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
Growing leaves on Nicotiana tabacum.L of the same ages were selected.The tobacco mosaic virus (6×10 -3 mg/mL) and inoculated on the whole leaves by dipping, 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 [10].For each compound, three repetitions were conducted to ensure the reliability of the results.

Figure 1 .
Figure 1.The molecular structure of compound 4l.

Figure 2 .
Figure 2. Packing diagram of the unit cell of compound 4l.

Table 1 .
Different reaction conditions used for the microwave irradiation synthesis of 4a.

Table 2 .
Yields of the title compounds

Table 4 .
The curative effects of the new compounds 4a-4l against TMV in vivo*.