One Pot Synthesis of α-Aminophosphonates Containing Bromo and 3,4,5-Trimethoxybenzyl Groups under Solvent-free Conditions

New α-aminophosphonates were synthesized by the Kabachnik-Fields reactionof 3,4,5-trimethoxybenzaldehyde (TMB) with p- or m-bromoaniline and a dialkylphosphite under solvent-free conditions. TMB was prepared from gallic acid via a fourstep synthetic sequence involving etherification, esterification, hydrazidation andpotassium ferricyanide oxidation. The structures of all synthesized compounds wereconfirmed by elemental analysis, IR, 1H-, 13C- and 31P-NMR spectral data. Compound 7gwas also characterized by X-ray crystallography. A half-leaf method was used todetermine the in vivo curative efficacy of the eight title products against tobacco mosaicvirus (TMV). It was found that compounds 7g and 7h possess good in vivo curativeeffects against TMV.


Introduction
α-Aminophosphonates, structural analogues of natural amino acids, have received wide attention in medicinal, bioorganic and organic chemistry.The applications of α-aminophosphonates have ranged from agriculture to medical uses as anti-cancer agents [1], enzyme inhibitors [2], peptide mimetics [3], antibiotics and pharmacological agents [4].To the best of our knowledge, only a few α-aminophosphonates containing bromo and 3,4,5-trimethoxybenzyl groups have been reported.As typical of a halogen, bromine has high electronegativity and is also known for its steric and lipophilic effects.As an active group, bromine is often introduced in the design of bioactive compounds.Many pesticides containing bromine are widely used commercially, and have a broad spectrum of activity, high efficiency and low toxicity, associated with their easy decomposition and minimal residues [5].
In view of all this information, a series of α-aminophosphonates containing bromo and 3,4,5trimethoxybenzyl groups were designed and synthesized in our laboratory for the purpose of exploring them as potential bioactive compounds.Substituted α-aminophosphonates are often synthesized in an organic solvent via a traditional Kabachnik-Fields reaction [10], but the use of organic solvents has a serious impact on the environment, whereas in contrast, solvent free reactions are benign and do not call for any drastic work-ups.
The TMB starting material 4 was prepared in turn in high yield from gallic acid (abundant in plant gallnut) in four steps: etherification, esterification, hydrazidation and potassium ferricyanide oxidation (Scheme 2).

Results and Discussion
A systematic study of the effect of reaction parameters on the process, including reaction temperature, the molar ratios of reagents and the amount of catalyst, was undertaken for optimization of the reaction.For this purpose, compound 7g was synthesized under different conditions.
The yield of the product was found to be significantly lower under otherwise similar conditions when no catalyst was used (Table 2, entry 2).The yield was improved as the amount of catalyst was increased from 5 mol% to 10 mol% (Table 2, entries 3, 4), but no noteworthy change was observed when the amount of catalyst used was increased to 15 mol% (Table 2, entry 5).Based on these results, the optimal conditions for the synthesis was established as the use of equivalent molar concentrations of the reactants and 10 mol% BF 3 •Et 2 O with a reaction temperature of 105~110 °C and a reaction time of 30 min.As may be seen from Table 3, using optimal conditions and with 10 mol% BF 3 .
Et 2 O catalyst, the compounds 7a-7h could be obtained in high yields (70.6-83.5%)after a much shorter reaction time (30 min).The compounds 7a-7h were obtained in 61.3-74.2%yield when the reaction was conducted for longer periods of time (2 h), but without the use of a catalyst.The structures of compounds 7a-7h were identified by elemental analysis, IR, 1 H-, 13 C-and 31 P-NMR spectral data.Taking compound 7g as an example, the absorption band at 3400 cm -1 (s) in its IR spectrum corresponds to an N-H stretch, the absorption bands at 1240 cm -1 (s) and 1001 cm -1 (s) correspond to P=O and P-O-C stretching, respectively.The 1 H-NMR spectra of 7g showed well-resolved doublets at δ 4.64 (J P-H = 24.05Hz) for the P(O)CH proton, while a singlet at δ 3.04 was due to the presence of the NH proton.In the 31 P-NMR spectra the phosphonate group resonance appeared at δp 22.69.The structure of compound 7g was definitively confirmed by X-ray diffraction analysis.

Antiviral activity bioassays
The results of the in vivo activity against TMV bioassays are given in Table 5. Ningnanmycin was used as reference antiviral agent.The data indicate that a change in the substituent might also affect the curative activity of title compounds 7a-7h.Compound 7h (R 1 = n-Pr, R 2 =3-Br) and compound 7g (R 1 = n-Pr, R 2 =4-Br) could cure TMV up to 54.5% and 44.3% at 500µg/mL.The other compounds all have relatively lower curative activity than 7h and 7g.

Conclusions
A series of α-aminophosphonates 7a-h containing bromo and 3,4,5-trimethoxybenzyl groups were synthesized by Kabachnik-Fields reaction under solvent-free condition.The procedure offers a great alternative to existing methodologies due to its ease of work up, faster reaction rates and high yields.The method is clean, free of any significant byproducts, environmental friendly and does not employ any solvent.A half-leaf method was used to determine the curative efficacy in vivo of the eight title products against tobacco mosaic virus (TMV).It was found that compound 7h had a good curative effect in vivo against TMV, with an inhibition rate of 54.5%.

General
Melting points (uncorrected) were measured on a XT-4 binocular microscope (Beijing Tech Instrument Co., China).The IR spectra (KBr disks) were recorded on a Bruker VECTOR 22 spectrometer. 1H-and 13 C-NMR spectra were determined at room temperature in the indicated solvents on a JEOL-ECX 500 NMR spectrometer (500 MHz for 1 H and 125 MHz for 13 C), with TMS as internal standard. 31P-NMR spectra were measured using as external reference an 85% H 3 PO 4 sample prepared by sealing a capillary containing 85% H 3 PO 4 in a 5 mm NMR tube containing a suitable amount of CDCl 3 for field locking.Elemental analysis was performed on an Elementar Vario-III CHN analyzer.The reagents were all of analytical reagent grade or chemically pure.All solvents were dried, deoxygenated and redistilled before use.TMB was prepared from gallic acid according to literature methods [11][12][13].O,O-Dialkyl phosphites were prepared as described in the literature [14].

General procedure for the preparation of products 7a-h
A mixture of 3,4,5-trimethoxybenzaldehyde (3 mmol), p-(or m-) bromoaniline (3 mmol) and dialkyl phosphite (3 mmol) was stirred in silicone oil bath at 108 °C for 2 h.The reaction was followed and monitored by TLC (petroleum ether-ethyl acetate=1:2 v/v).The resultant viscous liquid was dissolved in ether and then washed, first with saturated aqueous NaHCO 3 and next with distilled water.The organic layer was separated, followed by extraction of the aqueous layer with ethyl acetate (3×10 mL).The combined organic layers were dried over MgSO 4 and concentrated under reduced pressure.The residue was purified by recrystallization from petroleum ether to give compounds 7a-7h as white crystals.At the same time, compounds 7a-7h were also synthesized using BF  [16].Crystallographic data (excluding structure factors) for the structure have been deposited with the Cambridge Crystallographic Data Center as supplementary publication No. CCDC-634425.These data can be obtained free of charge from the CCDC via www.ccdc.cam.ac.uk/data_request/cif.

Antiviral Bioassay
Nicotiana tabacum.L leaves of the same age were selected.Whole leaves were dipped in a solution containing tobacco mosaic virus (6×10 -3 mg/mL), then the leaves were washed with water and dried.The test 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 [17].For each compound, three repetitions were carried out to ensure the reliability of the results.Inhibition rates were calculated using the expression:

Figure 2 .
Figure 2. Cell packing of the compound 7g.

Scheme 2 .
Synthesis of the starting material 4 from gallic acid.

Table 1 .
The influence of the molar ratio of reagents and the reaction temperature on the synthesis of 7a.

Table 2 .
The influence of the amount of catalyst BF 3 •Et 2 O on the synthesis of 7a.

Table 5 .
The curative effect of title compounds 7a-h at 500 µg/mL against TMV.