Synthesis of New 1,3,4-Thiadiazole and 1,2,3,4-Oxathiadiazole Derivatives from Carbohydrate Precursors and Study of Their Effect on Tyrosinase Enzyme

5-(1,2,3,4-Tetrahydroxybutyl)-2-methylfuran-3-carbohydrazide (2) was condensed with a variety of ketones to afford carbohydrazide derivatives 3–6. Acetylation of 3–5 afforded the acetyl derivatives 7–9, while periodate oxidation of 3–6 afforded the formyl derivatives 10–13. Acid catalyzed condensation of thiosemicarbazide or o-tolylthiosemicarbazide with the prepared aldehydes 10–12 gave thiosemicarbazone derivatives 14–19. Cyclization of the latter with acetic anhydride afforded 4,5-dihydro-1,3,4-thiadiazolyl derivatives 20–25. On the other hand, condensation of p-tosylhydrazine with the prepared aldehydes 10–12 afforded p-tosylhydrazone derivatives 26–28. Cyclization of 26–28 with acetic anhydride afforded 1,2,3,4-oxathiadiazole derivatives 29–31 respectively. Moreover, the obtained results regarding to the effect of some of the prepared compounds on tyrosinase enzyme showed that the majority of these compounds having an inhibitory effect; especially compounds 12, 16, 17, and 28.

In addition, tyrosinase is involved in dopamine biosynthesis, which has been shown to be involved in the control of movements, and the signaling of errors in the prediction of reward, motivation, and cognition. Cerebral dopamine depletion is the hallmark of Parkinson's disease [31]. Other pathological states have also been associated with dopamine dysfunction, such as schizophrenia, autism, and attention deficit hyperactivity disorder [32].
The structure of hydrazones 3-6 was proven by their 1 H-NMR spectra, which showed the NH proton as a singlet at δ 9.87-9.20, the proton at position-4 in the furan ring as a singlet at 7.27-6.57 ppm, the 1'-OH proton at 5.06-5.05 and the rest of the sugar protons at the 4.58-4.28 range. The methyl protons at position-2 in the furan ring appeared as a singlet at δ 2.44-2.21 ppm; additionally the disappearance of the two NH 2 protons was observed (see Experimental). It was observed that the C-1' hydroxyl proton of compounds 3-6, resonates at lower field (5.06-5.05 ppm) than the rest of the sugar protons. Electronic deshielding by the adjacent base residue undoubtedly is a major factor in causing these signals to appear at low field. Additional deshielding might also arise through the formation of an intramolecular hydrogen bond with the oxygen of the furan ring. Hydrogen bonding of this type was suggested in polyhydroxyalkyltriazole analogs [36] and polyhydroxyalkylpyrazolo-[3,4-b]-quinoxalines [37] having the D-arabino configuration of the side chain. In addition, the mass spectra of compounds 5 and 6, as examples of the series, showed the corresponding molecular ion peaks at m/z 377 and 342, respectively. On the other hand, acetylation of 3-5 afforded the corresponding acetyl derivatives 7-9 in 45-86% yield (Scheme 1). The 1 H-NMR spectra of compound 8 and 9 showed the disappearance of the OH protons in the sugar region, the O-acetyl protons at δ 2.43 and 2.23 ppm, respectively, and peaks at 2.59, 2.03 for the N-acetyl protons, respectively (for the other protons see the Experimental). Periodate oxidation of compounds 3-6 afforded the corresponding formyl derivatives 10-13, in 36-65% yields (Scheme 1). 1 H-NMR spectra of compounds 10-12 showed the NH proton as a singlet at δ 9.32, 8.91 and 9.36 ppm. respectively, and the formyl group proton as a singlet at δ 9.87, 9.34, and 9.44 ppm, respectively (for other protons see the Experimental). The mass spectrum of compound 13, showed the molecular ion peak at m/z 250, which was also the base peak. Acid catalyzed condensation of thiosemicarbazide or o-tolylthiosemicarbazide with the prepared formyl derivatives 10-12 gave thiosemicarbazone derivatives 14-19 in 43-99% yield (Scheme 1). The mass spectra of compounds 14, 16-18 showed the molecular ion peaks at m/z 343, 358, 433 and 449 respectively. Cyclization of the prepared compounds 14-19 with acetic anhydride afforded 1,3,4-thiadiazole derivatives 20-25 in 40-73% yield (Scheme 1).
The 1 H-NMR spectra of compounds 20-22 showed the disappearance of the NH 2 protons and CH=N proton. Instead, the N-Ac methyl protons appeared as a singlet. Interestingly, it was noted that the 1 H-NMR spectra of compounds 21 and 22 showed the proton at position-4 in the furan ring as a doublet signal at δ 6.69 and 7.22 instead of a singlet signal due to the long rang interaction between H-furan and the NH proton of the amide group. However a theoretical study of the NMR of compound 21 was attempted whereby the stable conformer of this compound was first established using the universal force field UFF molecular mechanics method ( Table 1). After that the {B3LYP/6-31G (d)} density functional approach was used to fine tune the geometry of the compound. The Orca computational chemistry program was used in this step. According to the calculation the distance between the NH proton and the H-furan is equal to 2.304 Å, which is the same value of the distance between the methylene protons and the methyl protons in the ethanol molecule. In the same way, H-furan appeared as a doublet due to the coupling interaction with the NH proton, while the proton of the NH group appears as a singlet, so the question is why the interaction with the H-furan didn't affect the signal of (NH) proton. This is attributed to the ionization factor [38] (Figures 1 and 2).   The mass spectra of compounds 20 and 22 showed the molecular ion peaks at m/z 427 and 526, respectively. The mass spectrum of compound 23 showed the molecular ion peak at m/z 560. In addition, condensation of p-tosylhydrazine with the formyl derivatives 10-12 afforded p-tosylhydrazone derivatives 26-28 respectively in 42-83% yield (Scheme 2). The 1 H-NMR spectra of compounds 26 and 27 showed the disappearance of the aldehyde proton. The two NH protons showed as a singlet at δ 9.36, 9.71, and 9.36, 10.47, respectively, the CH 3 protons of the p-tolyl moiety as a singlet at δ 2.36 and 2.32 ppm, respectively (see Experimental part). The mass spectra of compounds 26 and 27 showed the molecular ion peaks at m/z 438 and 454, respectively.
Similarly, cyclization of these hydrazones 26-28, with acetic anhydride afforded 1,2,3,4oxathiadiazole derivatives 29-31 in 32-54% yield (Scheme 2).The 1 H-NMR spectra of the compounds 29 and 31 showed the disappearance of both the CH=N and the NHSO 2 proton signals. The 1 H-NMR spectra showed the CH 3 protons of the p-tolyl group as a singlet at δ 2.40, 2.49 ppm, the CH 3 -C=N protons as a singlet at δ 2.05, 2.09 and the CH 3 -furan protons as a singlet at δ 2.40, 2.49, respectively. The mass spectra of compounds 29 and 30 showed the molecular ion peaks at m/z 420 and 478, respectively. Scheme 2. Synthesis of oxathiadiazole derivatives.

Biological Activity Assay
Tyrosinase was prepared from mushrooms in a phosphate buffer (50 mM, pH 6.0) according to the method of Yang and Robb [39], and the obtained supernatant after centrifugation was used as a source of enzyme.

Enzyme Activity Assay
The activity of the prepared enzyme solution was determined by following the formation of dopachrome spectrophotometrically at 30 °C, after addition of 50 μL enzyme preparation to a cuvette containing 1.2 mL phosphate buffer (50 mM, pH 6.0) and 0.8 mL L-Dopa (10 mM), the solution was immediately mixed and the increase in absorbance at 475 nm (indicating the formation of dopachrome) was recorded using UV-20100-spectrophotometer. Blank experiment was carried out as mentioned above using 50 μL of buffer instead of enzyme preparation [40].

Results
The obtained results showed that all these compounds are inhibitors for tyrosinase, except for compounds 18, 19 and 26 which were found to be activators of tyrosinase.

Type of Inhibition
The type of inhibition of N'

General Methods
Melting points were determined on a Koffler block and are uncorrected. IR spectra were recorded on Perkin Elmer 1600 USA Spectrometer. 1 H-NMR were 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 instrument. Elemental analysis was determined at the Regional Center for Mycology and Biotechnology, Al-Azhar University Plus. Optical rotation was obtained at 22 °C with a Perkin-Elmer model 241 Polarimeter equipped with a 10 cm, 1 mL micro cell. Thin layer chromatography (TLC) was carried out on silica gel plates. Solutions were evaporated under diminished pressure unless otherwise stated. The ChemDraw-Ultra-8.0 software has been used to name the prepared compounds.