Synthesis and Bioassay of a New Class of Furanyl-1,3,4-Oxadiazole Derivatives

Tyrosinase enzyme is a monophenol monoxygenase enzyme, which plays an important role in human as a rate limiting step enzyme for different specific metabolic pathways, as well as its useful application in industry and agriculture. So this study was carried out to test the effect of newly prepared compounds containing 1,3,4-oxadiazoles with different substituted groups on tyrosinase enzyme activity, hoping to use them in the treatment of some diseases arising from tyrosinase activity disorders such as Parkinson’s disease, schizophrenia, autism, attention deficit, hyperactivity disorder, and cancer.

The 1 H-NMR spectra of compounds 5a-c showed the disappearance of both the CH 3 and CH 2 protons of the ethyl ester group. Instead, they displayed two signals for the NH 2 protons in the δ 4.59-1.64range, and the NH proton of the hydrazide group at 9.82-9.00 ppm, respectively (for other protons see Experimental). The mass spectra of compounds 5a and 5c showed the molecular ion peaks at m/z 284 and 318, respectively. The base peaks of compound 5a and 5c appeared at m/z 171 and 105, respectively.
On the other hand, condensation of the hydrazide derivative 5a with a number of aldehydes afforded the corresponding hydrazone derivatives 6a-h (Scheme 1). The 1 H-NMR spectra of compounds 6a-h showed the disappearance of the NH 2 protons and instead, displayed a singlet signal for the CH=N protons in the δ 10.2-8.30 range, while the NH protons appeared as a singlet at δ 8.85, 9.98, 7.85, 8.80, 9.52, and 9.38, respectively (see Experimental). The mass spectra of the compounds 6d, 6e, 6f and 6h showed the molecular ion peaks at m/z 417, 393,440 and 447, respectively; while the base peaks appeared at m/z 176, 115, 77 and 358, respectively (see Experimental).
Cyclization of the hydrazones 6a, 6e, and 6f with acetic anhydride under reflux, afforded the oxadiazoline derivatives 7a-c (Scheme 1). The mechanism of their formation probably proceeded through the highly stable enolized form.

CAT
The 1 H-NMR spectra of compounds 7a-c showed the disappearance of both the NH and CH=N protons. Instead, their 1 H-NMR showed the methyl protons of the N-COCH 3 group as a singlet in the δ 1.24-1.22 ppm range (see Experimental). In addition, the proton at position-5 in the triazole ring (Compound 7c) resonates at lower field than the proton of the oxadiazolyl ring due to the strong electron attracting property of the triazole moiety. On the other hand, oxidative cyclization of the hydrazone compounds 6b and 6d with chloramine-T afforded the corresponding 1,3,4-oxadiazolederivatives 8a and 8b in high yield (Scheme 1). The 1 H-NMR spectra of compounds 8a and 8b showed the disappearance of both the NH and (CH=N protons (see Experimental). The mass spectrum of compound 8b showed the expected molecular ion peak at m/z 415, while the base peak appeared at m/z 91.

Enzyme Activity Assay
Tyrosinase enzyme was prepared from mushrooms in a phosphate buffer (50 mM, pH 6.0) according to the method of Yang and Robb [45]. The activity of the prepared enzyme solution was determined by following spectrophotometrically the formation of dopachrome at 30 °C. After addition of enzyme preparation (50 μL) to a cuvette containing phosphate buffer (1.2 mL, 50 mM, pH 6.0) and 10 mM L-dopa (0.8 mL), the solution was immediately mixed and the increase in absorbance at 475 nm (indicating the formation of dopachrome) was recorded with a UV-20100 spectrophotometer. A blank experiment was carried out as mentioned above using buffer (50 μL) instead of enzyme preparation [46].

Enzyme Activity Assay in the Presence of the Tested Compounds
Activity of the enzyme in the presence of the examined compounds was determined by following the above steps for the formation of dopachrome and each examined compound (0.8 mL, 10 mmol) separately, and the increase in absorbance at 475 nm was recorded, separately, as shown in Tables 1 and 2 and Figures 1 and 2. All tests carried out in triplicate.

Results
Our obtained data revealed that the examined compounds showed different effects [47] on the tyrosinase enzyme activity between inhibition and activation in which compounds containing one 1,3,4-oxadiazole ring in addition to the furan ring (compounds 6a, 6b, 6d, 6g) have an activating effect on the enzyme tyrosinase with different values (Table 1 and Figure 1). Compound 6a in which there is no substituent on the phenyl group, which probably makes it easier to bind with the active sites of the enzyme, showed the highest activation effect. The presences of substituents on the phenyl group such as halogens, or a nitro group (compounds 6b, 6d) decrease the activation ability of the compound, while compounds containing a CONHNH 2 group have inhibitory effects (5a, 5c).

General Methods
Melting points were determined on a Köfler block and are uncorrected. IR spectra were recorded on a Perkin Elmer 1600 spectrometer. 1 H-NMR was recorded on a JEOL JNM ECA 500 MHz instrument using tetramethylsilane as an internal standard. Mass spectra were recorded on a GC-MS solution DI Analysis Shimadzu Qp-2010 unit. Elemental analysis was determined at the Regional Center for Mycology and Biotechnology, Al-Azhar University. Thin layer chromatography (TLC) was carried out on silica gel plates. Solutions were evaporated under diminished pressure unless otherwise stated. The ChemDrew-Ultra-8.0 software was used for naming the prepared compounds.

Reaction of Compounds 6a, 6e, 6f with Acetic Anhydride
A mixture of 6a, 6e or 6f (100 mg, 27 mmol) and acetic anhydride (1 mL, 10.6 mmol) was refluxed for 15 min on gentle heating. The hot solution was poured onto ice water (10 mL) and the product which separated was filtered off, washed several times with water, recrystallized from ethanol and dried.