were easily obtained in 60–96% yields through addition-elimination condensation between compound 6 and different substituted
1
benzoyl hydrazine 7. Their structures were fully characterized by melting points, H NMR, 13C NMR, IR spectra and elemental
analysis (Supporting information).
In order to determine the stereo structure of the target compounds, a single crystal of representative compound 8g (R = 4-Br) was
prepared by slow evaporation of a solution of compound 8g in ethanol at 4 °C. As shown in Fig. 4, it gives a perspective view of 8g
with the atomic labeling system, and the result demonstrates the —CH=N—NH— bond bears an (E) rather than (Z) conformation.
Moreover, the crystal is distribution with dimer form and there is an intermolecular hydrogen bond between the two subunits. The
crystal conformation is more stable owing to the lower energy of the system. The crystal data for 8g: triclinic, a = 9.5767(5) Å, b =
9.6459(7) Å, c = 13.7997(9) Å, α = 84.771(6)°, β = 87.927(5)°, γ = 88.623(5)°, V = 1268.34(14) Å3, T = 104.6, space group P-1 (no. 2),
Z = 4, μ(Mo Kα) = 3.398 mm-1, Dcalc = 1.703 g/cm3, μ=3.398 mm-1,10125 reflections measured(6.08° ≤ 2Θ ≤ 52°), 4969 unique (Rint
= 0.0263) were used in all calculations. The final R1 was 0.0378 (I > 2σ(I)) and wR(F2) was 0.0935 (all data). Crystallographic data
have been deposited with the Cambridge Crystallographic Data Centre, and the deposition number was CCDC 1481606-1481607.
2.2. In vitro antifungal activity
All the target compounds were evaluated for their fungicidal activities against six plant fungal pathogens. The results were shown
in Table 1. The data suggested that all compounds had moderate to strong fungicidal activity. Compounds 8c, 8d, 8f, 8g, 8i, 8j, 8k, 8m,
and 8n exhibited more than 90% inhibition activities against V. mali, similar to Tiadinil (92.9%) at 50 µg/mL. Compound 8k also
displayed good activity against B. cinerea (87.5%), which is comparable with that of Tiadinil (80.4%) and Difenoconazole (92.1%).
Unfortunately, most compounds did not show better activities for the rest plant fungal pathogens compared with Tiadinil and
Difenoconazole. However, compounds 8k and 8n showed above 50% inhibition activity against other four fungi P. aphanidermatum, R.
solani, F. moniliforme and A. solani. These results indicated that the title compounds are exclusively efficient against V. mali mycelial
growth in vitro but not potent enough against the other five fungi. Thus, V. mali was chosen as the target for further study.
In order to intensively study the structure and activity relationship of the compounds against V. mali, therefore, different
concentrations of compounds 8a-8x were treated against V. mali. The EC50 values were calculated using linear-regression analysis, and
the results were shown in Table 1. Among these 24 compounds, 8d, 8f, 8g, 8i, 8j, and 8n showed lower EC50 values than the
commercial fungicide Tiadinil, especially 8g and 8n with the EC50 values of 1.6 and 1.9µg/mL, respectively, which were 4-fold higher
potency than Tiadinil. The results proved that introducing carbohydrazide hydrazone into the structure of Tiadinil to replace the amide
linkage is favorable to improve the fungicidal activity. However, the compounds showed weaker effectiveness than Difenoconazole.
Structure-activity relationship analysis generally indicates that: (i) The position of the substituent R on the benzene ring played an
important role in fungicidal activities: meta-position exhibited weaker activity than the para-position, however, better than the ortho-
position. Taking chloro substituents (8h-8j) as an instance, it is obvious that 8j (R=4-Cl, 2.8) < 8i (R=3-Cl, 3.8) < 8h (R=2-Cl, 19.4)
(―<‖ means inferior EC50 values). The 2, 5-dichloro substituent 8l (36.9 µg/mL) showed the poorest activity, which is even weaker than
8h. The 2, 4-dichloro substituent 8k (8.2µg/mL) showed the moderate activity, which is much higher than 8h and 8l, but lower than 8j.
The same rule can also be found in 8b−8d (−F), 8e−8g (−Br), 8m−8n (−CF3), 8o−8q (−NO2) and 8r−8s (−CH3) groups. (ii) The
electron-withdrawing groups are more favorable to the activity than the electron-donating groups in the same position. For example,
when substituent R is in the para position, compounds with the electron-withdrawing (8d, 8g, 8j, 8n, and 8q,) are more potent than
that with electron-donating substituent (8s-8u). Among of them, except 8q (4−NO2, 10.2 µg/mL) is close with 8s (4−CH3, 10.9 µg/mL)
and 8t (4−tBu, 9.4 µg/mL), and the other four compounds 8d (4−F, 7.1 µg/mL), 8g (4−Br, 1.6 µg/mL), 8j (4−Cl, 2.8 µg/mL) and 8n
(4−CF3, 1.9 µg/mL) are much higher than that of 8s and 8t. In conclusion, the compound containing halogen at para position exhibits
the best activity.
Different from most compounds with a very narrow spectrum against the fungi tested in Table 1, compound 8k showed broad
spectrum and thus was selected to determine the EC50 values against the six fungi. As illustrated in Table 2, 8k displayed different
levels activity against V. mali, B. cinerea, P. aphanidermatum, R. solani, F. moniliforme and A. solani with EC50 values of 8.20, 24.42,
15.80, 40.53, 41.48 and 34.16 µg/mL, respectively, which are still weaker than positive control except P. aphanidermatum.
2.3. In vivo antivirus activity
Curative activity of all title compounds against TMV in vivo was evaluated. The results were shown in Table 3, in which all the
compounds were active against TMV to some extent at the tested concentration. Among them, compounds 8a, 8b, 8e, 8j, 8m, 8o, and
8u presented moderate curative activity at 500 µg/mL, which were higher than that of standard Tiadinil. Especially, compounds 8a and
8b showed good anti-TMV activity with inhibition activity of 47.2% and 49.9%, respectively, which were close to the positive control
Ningnanmycin (57.9%). Interestingly, compounds containing R groups at ortho-position of phenyl have higher activity than those R
groups at meta-position. The results provide useful clues for further discovery of novel lead structures possessing good antivirus
activity.
3. Conclusion
A series of novel 4-methyl-1,2,3-thiadiazole-5-carboxaldehyde benzoyl hydrazone derivatives were designed and synthesized
through six steps with dimethyl carbonate as starting material. The bioassay indicated that the title compounds exhibited moderate to
strong antifungal activities against six fungi in vitro at 50 µg/mL and presented a certain degree of curative activity against TMV at