Synthesis and Biological Evaluation of S-Substituted Perhalo-2-nitrobuta-1 , 3-dienes as Novel Xanthine Oxidase , Tyrosinase , Elastase , and Neuraminidase Inhibitors

S-substituted perhalo-2-nitrobuta-1,3-dienes 3a, b were synthesized by the reaction of polyhalo-2-nitrobuta-1,3-dienes 1a, b with allyl mercaptan. 1-(2,3-Dibromopropanethio)-4-bromo-1,3,4-trichloro-2-nitrobuta-1,3-diene 4 was obtained from the addition of bromine to S-substituted polyhalo-2-nitrobuta-1,3-diene 3b in carbon tetrachloride. Sulfoxides 5a, b, and 6 were obtained from the reaction of thiosubstituted polyhalonitrobutadienes 3a, b, and 4 with m-CPBA in CHCl 3 . The structures of the new compounds were determined by spectroscopic data (FTIR, H NMR, C NMR, MS). These compounds exhibited antixanthine oxidase, antityrosinase, antielastase, and antineuraminidase activities.

Allylthiol derivatives, selected as the nucleophile, have diverse biological activities.These derivatives show antibacterial, anti-inflammatory, antithrombotic, anticancer, and antiatherosclerotic properties.Most of these effects are due to the SH group [20][21][22][23][24].At the same time, allylthio compounds are seen to decrease cholesterol and triglyceride levels in humans and animals [25,26].
Sulfoxide compounds, also synthesized in our work, have unique synthetic application to be used in synthetic studies.In the literature, these compounds have been termed as "chemical chameleons."They are used as synthetic mediators in the syntheses of several organic compounds having antiulcer, antibacterial, antifungal, and antihypertensive properties [27][28][29].
Xanthine oxidase (XO, EC: 1.1.3.22) is a complex metalloflavoprotein.This enzyme catalyzes the oxidation of hypoxanthine to xanthine and produces uric acid which leads to many diseases like gout and hyperuricemia [30].High XO activity leads to accumulation of the uric acid in blood.Hyperuricemia and gout diseases are risks of cardiovascular disease [31] and chronic kidney disease.Excessive uric acid levels in blood cause insulin resistance.Also, hyperuricemia is related to type 2 diabetes, metabolic syndrome, hypertension, kidney disease, hepatitis, inflammation, ischemiareperfusion, carcinogenesis, and ageing.XO is responsible for oxidative damage that causes many pathological diseases, such as gout, hyperuricemia, atherosclerosis [32], hepatitis, carcinogenesis [33], metabolic syndrome [34], vascular endothelium damage, and ageing.For this reason, inhibition of XO has been an obvious target to control uric acid level in blood.It has been shown that XO inhibitors may be useful for the treatment of hepatic disease, cancer, inflammation, and gout which is caused by the generation of uric acid and superoxide anion radical [35].
Tyrosinase (EC: 1.14.18.1) is a copper containing bifunctional enzyme.This enzyme is widely distributed in plants, microorganisms, fungi, and animals [36].Tyrosinase is also responsible for the production of neuromelanin.Excessive production of dopaquinone by oxidation of dopamine results in neuronal damage and cell death [37].For this reason, this enzyme contributed to the neurodegeneration associated with Parkinson's disease and other neurodegenerative disease processes [38].Also, cancers are characterized by abnormalities in tyrosinase activity [39].Tyrosinase is also associated with the host defense, wound healing, melting, and sclerotization of insects [40].Tyrosinase inhibitors were widely used in food, agriculture, and cosmetic industries and in the control of molting process of insects.Many natural and synthetic tyrosinase inhibitors have been reported such as ascorbic acid, arbutin, kojic acid, aromatic alcohols, azelaic acids, and aldehydes and polyphenol compounds such as resveratrol in recent years [41].However, most of them suffered from limitations such as low activity, high toxicity, and insufficient penetrative ability.Also, these substances have lower therapeutic effect, mutagenicity, and cytotoxicity potential.For this reason, the development of novel, potent nontoxic and stable tyrosinase inhibitors has a great importance in the medical, cosmetic, and agricultural fields.
Elastase (EC: 3.4.21.37) is a serine protease of the chymotrypsin family that hydrolytically degrades extracellular matrix components such as elastin, fibronectin, collagen, proteoglycans, laminin, and some matrix metalloproteinases [42].This enzyme is particularly abundant in the skin, arteries, lungs, and ligaments.Neutrophil elastase plays an important role in some processes like blood coagulation, apoptosis, and inflammation.Inhibition of the elastase activity could be used for protection against various diseases such as chronic obstructive pulmonary disease, cystic fibrosis, acute respiratory distress syndrome, acute lung injury, ischemia, and reperfusion injury [42].Neutrophil elastase also plays a main role in both acute pathogenesis and chronic functional recovery after traumatic brain injury [43].
Neuraminidase (NA, EC: 3.2.1.18)is one of the two glycoproteins on the surface of influenza virus.NA activity is increased in the serum patients and animal models of sepsis.This event indicates that NA is associated with the pathogenesis of inflammation during infection [44].NA is a key validated drug target in commercially available antiviral drugs.NA inhibitors, such as zanamivir, peramivir, and oseltamivir are commonly used for prevention and treatment of influenza [45].NA inhibitors are in wide use for the treatment of influenza [46].In the literature, thiourea and thiadiazole compounds have been reported to be effective against HIV and to have bacterial action [47].
Enzymes are biological macromolecules that accelerate or catalyze chemical reactions in biological systems.Some chemical, natural compounds and drugs alter normal enzyme activities by specific enzyme inhibition [48].
The increasing activities of enzyme cause different types of damage in the body.In this study, firstly we aimed to synthesize S-substituted perhalonitrobutadienes.The synthesized compounds were characterized by FTIR, 1 H NMR, 13 C NMR, MS.The second aim of this study is to investigate the xanthine oxidase, tyrosinase, elastase, and neuraminidase inhibition effect of these S-substituted perhalonitrobuta-1,3diene compounds.The inhibition of these enzymes is important for the treatment of many diseases such as gout, influenza, and cancer.
Compounds 3a, 3b, and 4 were subjected to oxidation with m-chloroperbenzoic acid in chloroform at 0 ∘ C to oxidize the thioether group.After the reactions, new sulfoxide derivatives 5a, 5b, and 6 were obtained (Scheme 1).
The compound 3b yielded 13 C-NMR peaks as the following: methylene carbon bound to sulfur at  = 37.7 ppm, CH= group for allyl substituent at  = 129 ppm, and the =CH 2 carbon at  = 120.4ppm.The compound 4, which is the bromine added product of 3b, yielded the following 13 C-NMR data: CH-Br carbon in the propyl group at  = 46.1 ppm and the CH 2 -Br carbon at  = 33.6 ppm.At the same time, 1 H-NMR spectrum shows the propyl group protons instead of the characteristic CH= group for allyl system and CH 2 = group.Methylene protons bound to sulfur, in compound 4, yielded 1 H-NMR peaks in the range  = 3.3-3.5 ppm, CH 2 protons bound to bromine atoms at  = 3.6-3.9,and CH proton at  = 4.2-4.3ppm.These data show that the double bond in the allyl group formed an added product with bromine.Meanwhile, in MS, the compound 4, which is an addition product, had a molecular ion peak at 531.3 [M + NH 4 ] + .Oxidation reactions are successful when the sulfoxide compounds 5a, 5b, and 6 are viewed in terms of spectroscopic Scheme 1: Synthesis of S-substituted perhalonitrobuta-1,3-diene compounds.
data.FTIR data of 5a and 5b showed characteristic S=O band at 1071 cm −1 , and FTIR data of 6 showed characteristic S=O band observed at 1073 cm −1 .Compound 5a yielded molecular ion peak at 326 [M + H] + in the mass spectrum, whereas compound 5b yielded molecular ion peak at 369.9 [M + H] + .When the mass spectrum of compound 6 is investigated, the ion peak is at 548.In the compound 4, there is a free sulfur group and this sulfur group has two unpaired electrons.Molybdenum is in the active center of XO.Molybdenum and sulfur atoms can be found in a complex and this complex formation shows inhibitory effects on XO.It has been pointed out in the literature that compounds with sulfur atoms in the structure exhibit xanthine oxidase inhibitor activity [60].It has been reported by Song et al. (2015) that febuxostat with sulfur in its structure inhibits the enzyme XO at a high rate.It was determined that the enzyme inhibited is similar to allopurinol in compound 4 which had a free sulfur group in its structure [61].
All compounds clearly showed a concentration dependent inhibition against tyrosinase activity (Table 2).Low IC 50 values indicate higher enzyme inhibitor activity.The results show that compounds 5b and 6 could inhibit tyrosinase.As shown in Table 2 the IC 50 values of 5b and 6 were similar.However, IC 50 values of 4 and 5a compounds were higher.Antityrosinase activity of S-substituted perhalonitrobuta-1,3diene compounds and standard decreased in the order of kojic acid > 6 > 5b > 4 > 5a.Sulfur containing compounds, such as thiourea, thiosemicarbazone, and thiocarbonyl compounds can form chelate with transition metal ions.In the literature, thiosemicarbazone derivatives had been reported to be efficient tyrosinase inhibitors [62].The inhibitory effect of thiosemicarbazone derivatives on tyrosinase exhibited potent inhibitory activity.The reason of this may be that the sulfur atom in the structure of the compound can chelate the two copper ions in the active side of enzyme.Many thiourea derivatives were known to be competitive inhibitors of tyrosinase due to their chelating ability to copper ions at the active sites [63].It was reported in the literature that sulfur atoms inhibit tyrosinase via bonding the copper ions in the active site of the enzyme [64].Liu et al. found that thiosemicarbazone derivatives showed high tyrosinase inhibition [65].
According to a previous study thio groups containing compounds act as o-dopaquinone scavengers [66].In our study, it can be claimed that sulfur atom which is found in compound 6 inhibited the enzyme by binding to the copper ion at the active site.Inhibition effects of elastase activities are shown in Table 3.The elastase inhibitor activities of S-substituted perhalonitrobuta-1,3-diene compounds were found to increase in a dose depending manner (Table 3).The inhibition was increased with increasing S-substituted perhalonitrobuta-1,3dienes concentration.Amongst the compounds, the best inhibition was found for the compound 4, followed by the 6, 5b, and 5a.A high elastase inhibition (58.55 ± 2.37%) was seen at 2000 M for 4 and its IC 50 value was 1476.79 ± 56.03 M.However, 5b and 6 showed nearly the same inhibition.The inhibitory potency of compounds was in the order of ursolic acid > 4 > 6 > 5b > 5a.In our study, it was seen that S-substituted perhalonitrobuta-1,3-diene compounds inhibit elastase in low ratio.It was reported by Stolk et al. [67] that thiol agent has an effect on elastase activity.In addition, Rodis and Digenis [68] reported that low molecular weight thiocarbamate compounds also have inhibitory effect on leukocyte elastase activity.Sokmen et al. showed that triazole compounds which include halogen, carboxylate, heterocyclic sulfide, sulfone, and/or methoxy groups in their structure inhibit elastase enzyme [42].In our study, it can be assumed that compounds which have halogen and sulfoxide groups in their structure inhibit elastase enzyme, yet, not in high ratio.
The neuraminidase inhibition activities of S-substituted perhalonitrobuta-1,3-diene compounds were given in Table 4. NA inhibitory activities of S-substituted perhalonitrobuta-1,3-diene compounds were found to increase in a dose dependent manner.Amongst them, the best inhibition was found for the compound 6.All compounds clearly showed a concentration dependent inhibition against neuraminidase activity.The inhibitory effect of compound 6 on neuraminidase activity was better than quercetin.Antineuraminidase activity of 4, 5a, 5b, and 6 decreased in the order of 6 > quercetin > 5b > 5a > 4. It was found that the compounds which have sulfoxide groups have higher NA inhibitory activity.A higher NA inhibitor activity is associated with a lower IC 50 value.
Compound 6 that contained halogen atoms and sulfoxide group showed excellent inhibitory activities.The inhibitory potential of compounds 5b and 5a was found greater than that of 4. Compound 6 demonstrated higher neuraminidase inhibition activity than that of quercetin (Table 4).A Ki value is the binding affinity constant of each inhibitor.Compound 6 was the best neuraminidase inhibitor (Ki = 108.37 ± 14.65 M).Quercetin showed a Ki value of 124.83 ± 14.95 M against neuraminidase activity.The inhibition mechanisms of compound 6 and quercetin were competitive.The quercetin which is a natural flavonoid has antiviral property [69][70][71].This substance was used as standard substance for antineuraminidase activity assay in our study.However, the synthesized compound 6 inhibited neuraminidase at higher rate than quercetin.It was declared that the flavonoid and phenolic compounds which are obtained from plants have antineuraminidase activity [72,73].The hydroxyl groups exist mostly in the structure of these compounds.There exists sulfur in the synthesized compounds in our study.Sulfoxides have antibacterial and antifungal activities [74].It can be assumed that the compound 6 which has sulfoxide groups has higher inhibition activity than the compounds that have hydroxyl groups.
This indicates that sulfoxide and halogens bind to active sites of NA enzyme.In our current study, the compound 6 which has sulfoxide and halogen groups in its structure has the highest antineuraminidase activity.Compound 6 could be a new lead for further modifications to discover more potent inhibitors.

Materials and Methods
3.1.General.All chemicals were obtained from commercial suppliers and without further purification.Thin layer chromatography was performed on Merck (60 F 254) TLC-plates (aluminum based).Purifications were carried out by means of column chromatography on silica gel 60 (Merck, 63-200 m particle size, 60-230 mesh).Melting points were measured on a Buchi B-540 apparatus and were uncorrected. 1H NMR and 13 C NMR spectra were recorded with Varian UNITY INOVA spectrometers with 500 MHz frequency for 1 H and 125 MHz frequency for 13 C NMR in ppm (). 1 H NMR spectra and 13 C NMR spectra in CDCl 3 refer to the solvent signal center at  7.2 and  77 ppm, respectively.IR spectra were recorded by ATR on Thermo Scientific Nicolet 6700 spectrometer.Mass spectra were recorded on Shimadzu LCMS-8030 triple quadrupole spectrometer in ESI (+) polarity.

General Procedure for the Synthesis of Sulfoxides 5a, 5b, 6.
Compound 3a, 3b, or 4 (1 mmol) in 30 mL of chloroform was mixed with m-chloroperbenzoic acid (4 mmol) in 30 mL of chloroform at 0 ∘ C for 24 h.The progress of the reaction was monitored by TLC.A solution of 2 M NaOH and chloroform was added to the reaction mixture.The organic layer was separated and dried with Na 2 SO 4 .The solvent was evaporated under low pressure.The product was purified by column chromatography.

1-(Allylsulfinyl)-4-bromo-1,3,4-trichloro-2-nitrobuta-1,3-diene (5b
where  represents the activity of the enzyme without sample and  is the activity of XO in the presence of the sample.Allopurinol was used as a standard compound as control.

Tyrosinase Inhibitory Activity.
Tyrosinase inhibitory activity was determined spectrophotometrically [76].Briefly, test reaction mixtures were prepared by adding mushroom tyrosinase, sample, L-tyrosine, and sodium phosphate buffer (pH 6.5).The resulting mixture was incubated for 10 mins at 37 ∘ C and absorption value was measured at 475 nm.The percentage of the inhibition of tyrosinase activity was calculated according to the following equation: where  represents the activity of the enzyme without sample and  is the activity of tyrosinase in the presence of the sample.Kojic acid was used as a standard compound as control.

Elastase Inhibitory Activity. Elastase inhibitory activity
was examined in this study by using N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide (STANA) as a substrate and by measuring the release of p-nitroanilide at 410 nm [77].The percentage of the inhibition of elastase was calculated according to the following equation: where  represents the activity of the enzyme without sample and  is the activity of elastase in the presence of the sample.Ursolic acid was used as a standard compound as control.
3.5.4.Neuraminidase Inhibitory Activity.NA inhibitory activity was determined by using the method described by Myers et al. [78] with some modification.The reaction was carried out with the enzyme (2.5 × 10 −3 U/mL), acetate buffer (pH: 5.0), and sample substrate (4-methylumbelliferyl-a-D-N-acetylneuraminic acid sodium salt hydrate, 0.125 mM) and was incubated for 10 minutes at 37 ∘ C.After reaction was stopped by adding glycine-NaOH buffer (pH: 10.4), the fluorescence of reactions was measured spectrofluorometrically.The emission wavelength was 440 nm, while excitation wavelength was 360 nm.The percentage of the inhibition was obtained by the equation below: Quercetin was applied as a standard compound as control.
The Ki values and inhibition types for compound 6 and quercetin were calculated by Lineweaver Burk curves for neuraminidase enzyme [79].
For all the enzyme inhibition, the results are given as half maximal inhibitory concentrations (IC 50 ) values, calculated from the regression equations prepared from the concentrations of samples.