Background

α-Amido sulfones are known for their wide range of application in asymmetric synthesis [1]. Different carbanionic nucleophiles can be reacted with α-amido sulfones, affording a wide variety of amino derivatives [2–7]. In addition, considerable research has been conducted on the reaction of nitrogen nucleophiles and α,β-unsaturated carbonyls [8–11]. Heteronucleophiles as well as carbanionic reagents can react with α-amido sulfones, thus giving the opportunity to prepare a large array of amino derivatives. Furthermore, a series of amino derivatives containing 1, 3, 4-oxadiazole ring were synthesized by the reaction of 1, 3, 4-oxadiazole-2-thiol with suitably substituted amines and formaldehyde in ethanol [12]. Based on these reports, we executed an inorganic base-mediated reaction of 5-subsititued phenyl-1, 3, 4-oxadiazole-2-thiols as the source of nitrogen nucleophiles with N-benzoyl-α-amido sulfones to produce N, N-aminals. Interestingly, the resulting compounds bearing a 1, 3, 4-oxadiazole ring are often associated with significant fungicidal and insecticidal activities [13–15]. Nevertheless, as of today, there has been no report on antiviral activities of N-substituted benzamides bearing 1,3,4-oxadiazol unit. We report synthetic and antiviral studies of the title compounds in the ensuing sections.

Results and Discussion

The synthetic route to the title N-substituted benzamides 5 is shown in Scheme 1. In order to prepare the key electrophilic component N-benzoyl-α-amido sulfones 4 for the final reaction, a three components reaction involving aromatic aldehyde, benzamide and sodium sulfonate was used by modifying the procedure reported by Chemla [16]. The reaction was fast, free of any significant side products formation and the pure product was isolated in moderate yield through recrystallization. The sulfones were characterized by 1H NMR, 13C NMR and IR spectral data. Finally, reaction of nitrogen nucleophiles 3 with N-benzoyl-α-amido sulfones in the presence of basic catalyst KOH in CH2Cl2 at room temperature afforded the desired N-substituted benzamides 5 bearing 1,3,4-oxadiazol group [Additional file 1]. The reaction conditions were optimized by taking compound 5a as the model. The effect of different solvents and bases was studied at room temperature with a fixed reaction time of 24 h (Table 1). Under these conditions, dichloromethane (CH2Cl2) provided the product in higher yield compared to toluene, tetrahydrofuran (THF) or acetonitrile. Amongst the various bases screened for the experiment, KOH gave the best result and higher conversion was achieved when 1.2 equiv of KOH was used instead of 1.0 equiv. Under optimized conditions, the isolated yield of the N-substituted benzamides 5a reached as high as 70% when the reaction mixture was stirred for 24 h in CH2Cl2 using 1.2 equiv of KOH.

Scheme 1
scheme 1

Synthetic sequence to N-substituted benzamide analogues 5 containing 1,3,4-oxadiazole ring.

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Table 1 Effect of different solvents and bases for the synthesis of 5aa
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Antiviral activity and structure-activity relationship

The results of in vivo antiviral activity studies of the N-substituted benzamides 5a-5u against CMV are given in Table 2. Ningnanmycin was used as the reference antiviral agent. Most of the compounds showed promising results in terms of curative bioactivities at 500 μg/mL. The comparison of the antiviral activity of the products with commercial reference leads to the following conclusions: (a) The antiviral activity is affected by the type of the substituents present in the compound. The compounds containing 2-fluorophenyl or 4-fluorophenyl group showed better anti-CMV activity compared with those derived from other groups. In particular, N-substituted benzamides 5o (R1 = o-F, R2 = p-Cl) and 5c (R1 = p-F, R2 = m-Cl) displayed moderate curative rates (48.7% and 42.2%, respectively) against CMV at the concentration of 500 μg/mL. These values were comparable to the curative rate (53.4%) shown by the commercial reference Ningnanmycin, and superior to other compounds bearing different substituents. (b) Substituents have a certain influence on the activity. Compared with the compounds 5o and 5c bearing suitably substituted aryl substituents, the compounds bearing an unsubstituted phenyl ring, such as 5a and 5n, showed lower inhibitory activities. (c) The structural modification caused by changing the substituents (R1 and R2) in the phenyl ring have a wide impact on anti-viral activity of the prepared compounds. Bioactivity of various compounds having the same substituents at different positions of the phenyl ring is various. Thus, amongst the compounds 5b, 5c, 5o and 5p containing the same substituents at different positions of the phenyl ring, the compound 5o carrying 4-chloro and 2-fluoro groups in their respective phenyl rings exhibited better bioactivity than others. Although these compounds, in general, exhibited slightly lower activity in comparison with the commercial reference Ningnanmycin at the concentration of 500 μg/mL, some suitably substituted N-substituted benzamides bearing 1,3,4-oxadiazol moiety showed favourable antiviral activity in the preliminary studies. Subtle structural variation might lead to enhancement of activity and should be the direction of future research.

Table 2 Curative effect of the title compounds 5 against CMV in vivo a
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Experimental

Chemistry

The melting points of the products were determined on an XT-4 binocular microscope (Beijing Tech Instrument Co., China) and were not corrected. The IR spectra were recorded on a Bruker VECTOR 22 spectrometer in a KBr disk. 1H NMR (500 MHz), 13C NMR (125 MHz) and 19F NMR (470 MHz) spectral analyses were performed on a JEOL-ECX 500 NMR spectrometer at room temperature using TMS as an internal standard and CDCl3 as the solvent. Elemental analysis was performed on an Elementar Vario-III CHN analyzer. Analytical TLC was performed on silica gel GF254. Column chromatographic purification was carried out using silica gel. All the solvents and materials were of analytical-grade. Intermediate 1, intermediate 2 and 5-subsititued phenyl-1,3,4-oxadiazole-2-thiol 3 were prepared according to the reported methods [17, 18] and used without further purifications [Additional file 2].

Antiviral biological assay

The leaves of Nicotiana tabacum L. inoculated with CMV were selected and grinded in phosphate buffer and filtered through double-layer pledget. The filtrate was centrifuged at 8000 g and the supernatant liquid was the crude extract of virus. The whole experiment was carried out at 4°C. Absorbance values were estimated at 260 nm using an ultraviolet spectro-photometer.

Preparation of medicaments

Tested compounds and 2% Ningnanmycin aqua used as a reference antiviral agent were first dissolved in minimum volume of N, N-dimethylformamide (DMF) and then diluted with distilled water containing 1% Tween 20 at 500 μg/mL concentration.

Curative effect of compounds against CMV in vivo

Growing leaves of Chenopodium amaranticolor of the same age were selected. Crude extracts of CMV were dipped and inoculated with a brush on the whole leaves, which were previously scattered with silicon carbide. The leaves were washed by water after inoculation for 0.5 h and then dried. The compound solution was smeared on the left side of leaves, and the solvent was smeared on the right side for control. All plants were cultivated at 28±1°C with an illumination of 10000 Lux. The local lesion numbers appearing 6-7 d after inoculation were counted. Three repetitions were conducted for each compounds. The inhibition rate of the compound was calculated according to the following formula (av denotes average):

Conclusion

We have demonstrated a general and practical route for the synthesis of the N-substituted benzamides bearing 1,3,4-oxadiazol moiety in the presence of an inorganic base as the catalyst. The reaction of 5-subsititued phenyl-1,3,4-oxadiazole-2-thiol which serves as the source of N-nucleophile with in situ generated protected imine from N-benzoyl-α-amido sulfones provides a ready access to a series of structurally diverse N, N-aminals. The antiviral tests indicated that some of the synthesized compounds possessed of moderately high curative activity against CMV. The structure of the target products needs to be optimized to enhance their antiviral activity. Further studies on mechanistic aspects, enantioselectivities and asymmetric variants of catalysts for this reaction are currently being investigated in our group.