Synthesis and Fungicidal Activity of Pyrazolecarboxamide Containing α-Aminoacetanilide Moiety

A series of pyrazolecarboxamides containing α-aminoacetanilide moiety (4) were synthesized. These compounds were characterized by IR, MS and H NMR. Their fugicidal activities against Rhizoctonia solani, Sclerotinia sclerotiorum, Monilinia fructicola and Alternaria brassicae were evaluated. The results show that compound 4l and 4p, which bear only one electron-donating group on the para-position of phenyl or benzyl group, possessed excellent inhibitory activities against the above four fungi.

4-Chloro-3-ethyl-1-methyl-5-pyrazole carboxylic acid was prepared according to literature 11 .The pyrazole carboxylic acid (8.6 g, 0.0456 mol) and 2 drops of DMF was dissolved in toluene (60 mL), stirred at room temperature, thionyl chloride (8.0 mL, 0.1013 mol) was added dropwise in 1 h and the resultant reaction mixture was stirred for 6 h at 60 o C.After removal of the solvent and excess of thionyl chloride under reduced pressure, a viscous liquid residual was collected as the chloride compound (1) (8.85 g, 93.8%).

General procedure for the synthesis of 4a-p
The synthesis method was exemplified by the preparation of compound 4a.Compound 3 (1.0 g, 0.004 mol), o-chloroaniline (0.51 g, 0.004 mol), N, N'-dicyclo -hexyl carbodiimide (DCC, 0.99 g, 0.0048 mol) and 15 mL CH 2 Cl 2 was added successively into 100 mL three necked round bottom flask.After stirring for 15 min in cold water bath (0∼5 o C), 4-dimethylaminepyridine (DMAP, 0.05 g, 0.0004 mol) was added.Then the mixture was warmed to room temperature and stirred for 4 h.After filtration of insoluble material from the mixture, the solution was washed with 15 mL 5% NaOH, 15 mL 5% HCl, 15 mL 20% brine.The solution was dried over magnesium sulphate and the solvent was evaporated under reduced pressure.Compound 4a was obtained as white solid.After recrystallization in ethanol, pure white 4a (1.10 g, 77.6%) was provided.Others compounds were synthesized by the way similar to compound 4a.

Antifungal activities Radial fungal-growth assay
The antifungal activity was tested in vitro on Rhizoctonia solani, Sclerotinia sclerotiorum, Monilinia fructicola and Alternaria brassicae.The activities of the compounds were assayed as growth inhibition.Each compound was dissolved in acetone plus Tween 80 (1.0%).Equal volumes of acetone containing tested compounds were added to sterile cool agar media (Potato Dextrose Agar Difco) to give final 50 mg/L concentration for each substance.The final acetone concentration did not exceed 1.0% of the final volume in both control and treated cultures.Compound-amended agar medium was dispersed aseptically onto 9 cm diameter plastic Petri dishes (10 mL/dish).Each dish was inoculated with a 0.5 cm diameter mycelial disc of actively growing colonies.Three replicates were used for each compound, together with controls containing toxicant-free medium.The growth inhibition was calculated from mean differences between treated and control cultures as a percentage of the latter.The results were compared with standard fungicide Carbendazim.The growth was determined after 2 days of incubation for Rhizoctonia solani and Sclerotinia sclerotiorum, 4 days for Monilinia fructicola and Alternaria brassicae.

Synthesis
For the synthesis of compound 2, which was prepared by the amination of acyl chloride (1), the temperature should be controlled around 0 0 C. When the temperature was higher, the side reaction of amination between glycinate would take place, leading to polymerization 12 , that is, the amino group of one molecule ethyl glycinate could attack the ester group of another molecule of ethyl glycinate.Two different strategies were applied to obtain the series of title compounds (4a-4p); the first one involved the acyl chloride of compound 3 with different anilines, however, there was side reaction between the acyl chloride group of compound 3 and the amine group on itself, moreover, the side product was hard to be removed; the second one, which was applied by this paper, exploited the DCC/DMAP system.DCC can transform the acid into corresponding anhydride and DMAP can efficiently promote acylation of aniline with acid anhydrides to give the corresponding amides in good yields 13 .
The 1 H-NMR signals of the two NH units in title compounds were variable, which on the one hand, correlate with the chemical structure of the phenyl group connected to NH, on the other hand were strongly influenced by hydrogen bonding.It is well known that the more hydrogen bonding there is, the more the proton is deshielded and the higher its chemical shift will be.However, since the amount of hydrogen bonding is susceptible to factors such as solvation, concentration and temperature, so the chemical shift of NH units were not the same for all title compounds.

Fungicidal activities
The results of the in vitro fungistatic activity of compounds 4a-4p and commercial fungicide Carbendazim at 50 mg/L against fungi Rhizoctonia solani, Sclerotinia sclerotiorum, Monilinia fructicola and Alternaria brassicae are listed in Table 1, in which the fungistatic activity was expressed as inhibition percentage.The results in Table 2 showed that the synthesized target compounds were fairly active against nearly all four fungi to some extent.Compared to the control Carbendazim (58.7%), some of the compounds showed significant activities against Sclerotinia sclerotiorum, for example, the inhibition rates compounds 4c, 4f, 4l and 4p were all above 50% at 50 mg/L.Particularly, for compound 4l, the inhibition rate on Sclerotinia sclerotiorum was above 80%.Furthermore, compounds 4l and 4p, exhibited very strong inhibition against the other three fungi.The common structural feature of both was that the benzene ring or benzyl group (4l) was substituted with only one strong electron-donating substitute at site-4.Furthermore, when R was benzyl group (viz 4l) instead of a phenyl group, there was a dramatic increase in the activity, which suggests that the substitute on the amino moiety likely plays a positive role in the mechanism of their antifungal action.

Conclusion
On the whole, most of the title compounds exhibited good fungicidal activity.Hence, title compound could be useful for further optimization work in finding the potential antifungal compounds.

Table 1 .
Antifungal activity of title compounds