Synthesis, Structure and Antifungal Activity of New 3-[(5-Aryl-1,3,4-oxadiazol-2-yl)methyl]benzo[d]thiazol-2(3H)-ones

A series of new 3-[(5-aryl-1,3,4-oxadiazol-2-yl)methy])benzo[d]thiazol-2(3H)-ones were synthesized by reaction of (5-substituted-2-oxobenzothiazolin-3-yl)-acetohydrazide with various aromatic acids in POCl3 under reflux conditions. The structures of the title compounds were confirmed by 1H-NMR, 13C-NMR, IR, MS and elemental analysis. Furthermore, the structure of compound 4i was determined by single-crystal X-ray diffraction. The preliminary bioassy results indicated that some of them showed moderate inhibition activity against Colletotrichum orbiculare, Botrytis cinerea and Rhizoctonia solani.


Results and Discussion
The synthetic route to the title compounds 4a-4v is shown in Scheme 1. Substituted 2(3H)-benzothiazolones 1 were obtained by the cyclization of 4-substituted-2aminothiophenol with triphosgene, which was seldom reported. The reaction of 1 with ethyl chloroacetate in the presence of potassium carbonate gave the ethyl 2-(2-oxobenzothiazolin-3-yl)acetates 2. Their acetohyrazides 3 were produced by reaction of compounds 2 and hydrazine hydrate. Finally, the title 3-[(5-aryl-1,3,4-oxadiazol-2-yl)methyl]benzo[d]thiazol-2(3H)-ones 4a-v were obtained in 69.0-93.2% yields by refluxing acetohydrazides 3 and an aromatic acid in POCl 3 . In general, the yields of compounds 4 bearing chloro in the 5-position are lower than those possessing a hydrogen at that position. Their structures were confirmed by 1 H-NMR, 13 C-NMR, IR, MS and elemental analysis. In the 1 H-NMR spectra of title compounds 4, the peaks of the 3-position methylene groups appear in the δ 5.41-5.51 ppm range, and in the corrsponding 13 C-NMR spectra, they appear in the δ 36.67-37.22 ppm range. In the IR spectra of compounds 4a-v, the characteristic ν (C=O) stretching vibration signals appear at 1667-1703 cm −1 . Meanwhile, all the title compounds exhibited M + or [M+1] + peaks in the MS.
When compound 4i was recrystallized by slow evaporation from acetone, a single crystal was obtained and analyzed by X-ray diffraction crystallography. The molecular structure of compound 4i is shown in Figure 1 and the packing of the molecule in crystal lattice is illustrated in Figure 2. Its crystal structure is of monoclinic system, space group C2/c with a = 2.718 (2) nm, b = 1.2432 (10) nm, c = 0.9425 (8) nm, α = 90°, β = 108.666 (14)°, γ = 90°, V = 3.017 (4) nm 3 , Z = 8. The bond length of N(2)-N(3) is 0.1420 nm, which is shorter than the normal single N-N bond length (0.1450 nm). The bond lengths are 0.1388 nm and 0.1285 nm for N(1)-C (7) and N(2)-C(9), respectively, which are shorter than the normal single N-C bond length (0.1470 nm) and hence indicative of some double bond character. In the molecular structure of 4i, the CH 2 group is nearly perpendicular to the phenyl ring and oxadiazole ring with a θ angle of 110.5°, The oxadiazole ring (O2, C9, N2, N3, C10), phenyl ring (C11, C12, C13, C14, C15, C16), and benzothiazole (C1, S1, C2, C3, C4, C5, C6, C7, N1) are fairly planar with plane equation 4.260x + 8.778y + 5.694z = 4.311, 4.43x + 7.23y + 6.623z = 4.434, 26.998x − 0.047y − 4.027z = −1.253, and the largest deviations from the least squares plane are 0.0037 nm, 0.0054 nm, 0.0189 nm. Meanwhile, the oxadiazole ring is perpendicular to the benzothiazole ring about an angle of 86.1°, and nearly planar with the phenyl ring with an angle of 9.4°.  The in vitro antifungal activities of 4a-v against Colletotrichum orbiculare, Botrytis cinerea and Rhizoctonia solani at the dosage of 50 μg/mL were evaluated compared with the commercial fungicide propiconazole. The antifungal activity data are listed in Table 1. The preliminary bioassay results showed that all compounds exhibited certain inhibitory activity against all the tested fungi, and some of them possessed moderate antifungal activity. For example, compounds 4d, 4l, 4n, 4s and 4u exhibited more than 50% inhibitory activity against C. orbiculare, and compounds 4a, 4k, 4l and 4q displayed greater than 50% inhibition activity against R. solani, but their activities were still lower than that of the control fungicide (propiconazole), while most of compounds 4a-v showed activities against B. cinerea higher than propiconazole, especially compounds 4i, 4l, 4m, 4n, 4o and 4u showed above 60% inhibition activity against B. cinerea. The activity data indicated that compounds 4a-v did not exhibit improved inhibition when a chlorine atom was introduced in 5-position. In general, the compounds bearing an electron-withdrawing group (e.g., halogen and nitro) or methoxy on the aromatic ring (Ar), exhibited higher activity against B. cinerea than those bearing an alkyl substituent. It is also note worthy that the inhibition rates of 4a-v against R. solani evidently went up when a fluorine atom was introduced on the aromatic ring, especially in the para-position (i.e., compound 4l, which exhibited 76.37% inhibitory activity). Further studies on structural optimization and structure-activity relationships of these title compounds are in progress.

Materials and Reagents
Melting points were determined using an X-4 apparatus without calibration. 1 H-NMR and 13 C-NMR spectra were measured on a Bruker ADVANCE III instrument (500 MHz) using TMS as an internal standard and CDCl 3 or DMSO-d 6 as solvents. IR spectra were obtained on a Thermo Nicolet AVATAR 370 FT-IR instrument with KBr plates. Mass spectra were recorded on a Thermo Scientific ITQ 1100TM (EI) or Thermo-Finnigan LCQ-Advantage (ESI) instruments. Elemental analyses were performed on a Vario EL elemental analyzer. X-ray diffraction crystallography was measured on Rigaku Saturn 724 diffractometer. The reaction progress was monitored by TLC plates running in a PE-EtOAc solvent system, and spots were visualized by exposure to UV light (254 nm). All chemical reagents and solvents used in this study were commercial and were used without further purification.

Crystal Structure Determination
The crystal of compound 4i with dimensions of 0.20 mm × 0.18 mm × 0.16 mm was mounted on a Rigaku Saturn 724 diffractometer with a graphite-monochromated MoKα radiation (λ = 0.71073 Å) by using a Phi scan modes at 113 (2) K in the reange of 1.82° ≤ θ ≤ 25.02°. A total of 12122 reflections were collected, of which 2669 were independent (Rint = 0.127) and 2406 were observed with I > 2σ(I).
The calculations were performed with SHELXS-97 program [25] and the empirical absorption corrections were applied to all intensity data. The non-hydrogen atoms were refined anisotropically. The hydrogen atoms were determined with theoretical calculations and refined isotropically. CCDC No. 856797 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; Fax: +44 1223 336033; E-Mail: deposit@ccdc.cam.ac.uk).

Antifungal Activity Assays
The in vitro antifungal activities of 4a-v against Colletotrichum orbiculare, Botrytis cinerea and Rhizoctonia solani were evaluated using the mycelium growth rate test [26]. The method for testing the primary biological activity was performed in an isolated culture. Under sterile conditions, sample (1 mL) was added to the culture plates, followed by the addition of culture medium (9 mL). The final mass concentration was 50 μg/mL. Circle mycelium with a diameter of 4 mm was cut using a drill. The culture plates were cultivated at 24 ± 1 °C. The extended diameters of the circle mycelium were measured after 72 h. Propiconazole, a commercial fungicide, was used as a control, and sterile water was used as a blank. Three replications were performed. The relative inhibition rate of the circle mycelium compared to blank assay was calculated via the following equation: Relative inhibition rate (%) = [(dex − dex')/dex] × 100% where dex is the extended diameter of the circle mycelium during the blank assay; and dex' is the extended diameter of the circle mycelium during testing.