Synthesis and Evaluation of Some Sulfonamide-Substituted of 1,3,5-Triphenyl Pyrazoline Derivatives as Tyrosinase Enzyme Inhibitors

Pyrazoline is well-known as heterocyclic compound that can exhibit many biological effects. In this work, we synthesized a series of sulfonamide-substituted 1,3,5-triphenyl pyrazoline compounds as a promising tyrosinase inhibitor agent. These compounds prepared by multicomponent reaction of corresponding aldehyde, ketone, and hydrazine using sealed-vessel reactor. Pyrazoline compounds were tested for their tyrosinase inhibitor activity through in vitro assay. The test result found that compounds 4c, 4d, and 4e possessed better tyrosinase inhibitory activity compared to the reference inhibitor kojic acid. Compound 4c exhibited the strongest tyrosinase inhibitory effect with an IC 50 value of 30.14 µM. The results suggested that hydroxyl and methoxy substituents at para position are preferable. Furthermore, molecular docking studies result match the pattern of in vitro assay where the compound will provide a stronger binding interaction and lower binding free energies.


INTRODUCTION
Tyrosinase enzyme is a copper-containing oxidase which acts as a limiting enzyme in the synthesis of pigments such as melanin and polyphenol compounds (Di Petrillo et al., 2016) Melanin is a pigment that is responsible for the skin, hair and eyes color of humans and animals, which plays a key function in UV protection and is an essential defensive mechanism of the skin against any harmful factors (Pillaiyar et al., 2017;Qin et al. 2015). However, in human the overproduction of melanin in the skin due to tyrosinase overexpression can cause hyperpigmentation consequences such as freckles, melasma, age spots, and melanoma (Cui et al., 2018) Furthermore, tyrosinase also catalyzes the neuromelanin biosynthesis pathway, in which dopamine is oxidized to form dopaquinones. Excessive production of dopaquinones, on the other hand, causes neuronal damage and cell death. This showed that tyrosinase may be involved in the formation of neuromelanin in the human brain, as well as the neurodegeneration associated with Parkinson's and Huntington's disorders (Hasegawa, 2010;Pillaiyar et al., 2017). In terms of agriculture, overproduction of tyrosinase has been associated to the browning of a variety of fruits and vegetables, as well as alterations in their flavor and nutritional value, all of which can reduce their economic worth (Loizzo et al., 2012). Since tyrosinase is the limiting step enzyme in melanogenesis, tyrosinase inhibitors have become essential as depigmenting treatments in hyperpigmentation diseases.
Numerous synthetic tyrosinase inhibitors have been reported, including hydroquinone and kojic acid, which is frequently used in cosmetics to lighten the skin (Chang, 2009). However, there have been some major adverse effects linked to the usage of this chemical. Kojic acid has been linked to contact dermatitis and photosensitivity, while hydroquinone has been linked to exogenous ochronotic, contact dermatitis, corneal melanosis, nail hyperpigmentation, conjunctival hyperpigmentation as well as severe side effects includes peripheral neuropathy, fish odor syndrome, fetal growth retardation (Pollock et al., 2020). Therefore, further studies need to be done to find novel and biocompatible tyrosinase inhibitors that may be employed in the cosmetic and food sectors as whitening and anti-browning agents. Synthesized compounds such as aurones, chalcones, flavanones, and pyrazole derivatives have gotten a lot of interest as tyrosinase inhibitors. Specifically, pyrazoline derivatives because of its ability to interact with the hydrophobic protein pocket around the binuclear copper active site of tyrosinase (Qin et al., 2015).
Pyrazoline are mono-unsaturated 5-membered heterocyclic compounds containing three carbon atoms and two adjacent nitrogen within the ring. This compound has been reported to have many biological effects such as anticancer, antidiabetic, antidengue virus, antituberculosis (Ahmad et al., 2016;Herfindo et al., 2020;Jasril et al., 2019;Zamri et al., 2019) and pyrazolines with sulfonamide group also exhibit biological activity as urease inhibitor, which makes this compound interesting (Mojzych et al., 2017). Additionally, sulfonamides are a valuable therapeutic class. It exhibits antibacterial, hypoglycemic, highceiling diuretic, antithyroid, antiglaucoma properties, and anti-inflammatory (Mojzych et al., 2014). Moreover, a variety of sulfonamides have been reported to act as tyrosinase inhibitor (Lolak et al., 2020;Mojzych et al., 2014;Rahayu et al., 2022). Based on these findings, we explored the tyrosinase enzyme inhibitory activity of sulfonamide-substituted of 1,3,5-triphenyl pyrazoline. The hydroxyl group was also introduced to mimics kojic acid. Then the biological activity of the compounds was compared by molecular docking study and in vitro assay to tyrosinase enzyme.

Synthesis of 4-Hydrazinylbenzenesulfonamide (3)
Compounds 3 as one of starting material for this study was prepared according to previously reported literature (Rahayu et al., 2022).

Molecular Docking Studies
Molecular docking was performed to predict plausible binding mode of synthesized compound in the active site of tyrosinase. In this study, docking was performed using MOE 2020.0901 (Chemical computing group) software package. All synthesized compound structures were sketched using ChemDraw 17 for then these compounds were converted into 3D structure and minimized their energy with the protocol set up MMF94x as selected force field. Protein preparation consisted of some steps, it was begun with download the protein from the protein databank with PDB ID 2Y9X (www.rcsb.org), followed with water removed, alpha carbon and backbone atom minimized. Amino acid residues that have interacted with the native ligand were noted and keep in 2D. Furthermore, chain A was prepared in MOE 2020.0901 using the following steps. It was begun with deleted the ligand followed with selected the forcefield (CHARMM27). QuickPrep tools in MOE 2020.0901 was used for the protein preparation. Finally, this protein was saved in PDB format for then it was ready used as receptor. Active site of the protein was observed using site finder. Re-docking was performed with the placement and refinement of 50 and 10, respectively. The best spatial arrangement of the re-docked ligand was chosen with RMSD value less than 2 with the same interaction between re-docked ligand and the native ligand.

Mushroom Tyrosinase Enzyme Inhibition Assay
The pyrazoline compounds tyrosinase inhibitory activity were measured using a previously published method with minor adjustments (Cui et al., 2018). The substrates in this test were L-tyrosine. In a 96-well microtiter plate, 40 microliters of L-tyrosine (10 mM) were combined with 80 microliters of phosphate buffer (0.1 M, pH 6.8) and incubated for 10 minutes at 37 °C. Each well on the plate was then filled with 40 microliters of pyrazoline compounds (500, 250, 125, 61.5, 31.125 g/mL in 50% DMSO) and 40 microliters of mushroom tyrosinase (250 U/mL, in PBS), and the absorbance characteristics of the resulting mixtures were measured at 475 nm using a microplate reader. PBS was utilized as a blank control instead of the test sample, and kojic acid (50 µg/mL) was employed as a positive control. For each enzyme assay, the inhibition was calculated as follows: Each experiment was performed in triplicate (n=3). The IC50 value was measured by using nonlinear regression analysis of the dose-response curves.

RESULTS AND DISCUSSION
Sulfonamide-substituted 1,3,5-triphenyl pyrazoline 4a-f have been obtained from the one-pot reaction between aromatics aldehydes, aromatics ketones, and hydrazine derivate by using a closed-vessel reactor. The synthetic pathway for the desired compounds is depicted in Figure 1. In general, pyrazoline formation reactions can be carried out under acidic and basic conditions (Farooq & Ngaini, 2020). In this study, the reaction was carried out in presence of sodium hydroxide which gave good yield. As consequence, pyrazoline is formed through the formation of chalcone as an intermediate, followed by the formation of a pyrazoline ring by a cyclo-condensation reaction with hydrazine.
The compounds of 4a-c have different in position of hydroxyl group in the aromatic ring. In this case, 4b and 4c showed better yields than 4a because the hydroxyl group in meta (4b) and para (4d) make them avoid from unwanted intramolecular cyclization. This unwanted reaction can be observed when we used 2'hydroxyacetophenone as starting material (Zamri et al., 2016). The ortho position of hydroxyl group at starting material of 4a is possible to lead by product formation and reduce the possibility the reaction between the chalcone intermediate and another starting material (4-hydrazinylbenzenesulfonamide). So that, the compound 4a is obtained in low yield.
In case of compounds 4d-f, although the hydroxyl groups in acetophenones as their starting materials are in same position (para), there are differences in the type and position of substituents of benzaldehydes as their starting materials. The presence of two substituents (4-hydroxy and 3-methoxy) at benzaldehyde as starting material of 4d suspected of causing steric hindrance that can reduce the yield. Then, compound 4f with the higher yield than compounds 4c dan 4e were caused the presence of methoxy group in ortho position of starting material of 4f that given a bigger impact from negative induction effect, so that the carbonyl group of benzaldehyde as starting material of 4f become more reactive that the other monomethoxy benzaldehydes (meta and para). Another researcher has also reported the similar phenomena that the presence of monomethoxy substituent in ortho position of the benzaldehyde as starting material is also observed to give higher yield than meta and para (Dona et al., 2022).
The formation of pyrazoline compounds were confirmed by spectroscopic analyses. The FTIR spectra of 4a-f show the similarity in absorption pattern. Two absorption bands around ῡ 3386 and 3275 cm -1 show the presence of asymmetrical and symmetrical stretching of the N-H in the sulfonamide (SO2NH2) group, while the absorption at the range ῡ 1591 -1601 cm -1 show the presence of C=N stretch and the absorptions around ῡ 1335-1320 cm -1 indicate the stretching vibration of the C-N bond on the pyrazoline ring. In addition, the absorptions around ῡ 3075-3017 and ῡ 1516-1502 cm -1 indicate the presence of aromatic rings. Then, the broad absorption bands around ῡ 3273-3211 cm -1 indicate the stretching vibration of phenolic O-H bond. Furthermore, absorption bands around ῡ 1256-1238 and ῡ 1156-1150 cm -1 indicate the vibration of the C-O bond from the methoxy substituent and the S=O bond of sulfonamide group, respectively. 1 H NMR spectra of compounds 4a-f emphasize the formation of pyrazoline ring, by the appearance of the ABX protons pattern in the upfield region, as shown in Figure 2. In this case, the protons HA and HB are in the geminal position of the methylene carbon of the pyrazoline ring, while HX is a proton from the methine carbon of the pyrazoline ring. The peaks of protons HA appear at range δH 3.01 -3.29 ppm (dd, 1H), protons HB appear at range δH 3.85 -4.05 ppm (dd, 1H), and protons HX appear at range δH 5. 40 -5.56 ppm (dd, 1H). The difference in chemical shift between the two methylene protons (HA and HB) is due to the anisotropic effect of the bonding atom to the chiral carbon atom.
Each ABX peak appears as double of doublet (dd) with the difference in their coupling constants, where the proton HA has geminal coupling with proton HB and vicinal coupling with proton Hx (JAB = 17.4 -18.0 Hz and JAX = 4.8 -5.4 Hz), proton HB has geminal coupling with proton Ha and vicinal coupling with proton Hx (JBA = 17.4 -18.0 Hz and JBX = 11.9 -12.4 Hz), whereas proton Hx showed the vicinal coupling with proton HA and HB (JXA = 4.8 -5.4 Hz and JXB = 11.9 -12.4 Hz). The 1 H NMR spectra also showed a singlet peak of phenolic OH proton at the range δH 10.56 -9.60 ppm (s, 1H), NH2 protons of sulfonamide at the range δH 7.04 -6.98 ppm (s, 2H), aromatic protons at the range δH 7.67 -6.56 ppm, and methoxy protons at the range δH 3.83-3.69 ppm (s, 3H). Overall, the 1 H NMR spectra of synthesized pyrazolines has accordance between the number of protons of the obtained compounds and the target molecules. In this case, there are 21 protons in each of pyrazoline compounds.   Finally, the formations of target molecules were also confirmed through mass spectroscopic data. The molecular weight of each compound was calculated as [M+H] + and the molecular ion was found at their corresponding mass with high intensity. In addition, there are only a slight difference between calculated and measured masses. The differences are around 0.0002 -0.0021 m/z unit. Overall, the difference in masses were still less than 0.0030 m/z unit. Thus, they are still acceptable for compound with molecular weight less than 1000.

Tyrosinase Inhibitory Activity
In this work, the biological effect of pyrazoline compounds was evaluated. The monophenolase activity assay was used to test the tyrosinase inhibitory activity of these compounds. The reference drug in this experiment was kojic acid. Kojic acid is a fungus metabolite with inhibitory effect against tyrosinase. The ability of kojic acid to chelate copper at the active site of this enzyme has been attributed to its inhibitory effects in this context (Qin et al., 2015). As a result, kojic acid is frequently utilized as a positive control while searching for novel tyrosinase inhibitors. The effects of compound 4c on mushroom tyrosinase monophenolase activity were investigated using Ltyrosine as a substrate and kojic acid as a control. Initially, three compounds (4a-c) were screened for their inhibitory activity. The results found that 4c had the significant effect to the enzyme with IC50 value of 30.14 µM. Hydroxyl substituent of ring A at ortho and meta position is not preferable whereas the IC50 values of 4a and 4b were ˃ 100 µM (Table 1). Furthermore, 4c activity was compared to pyrazoline with different substituent position at ring B (4d-f). Similar pattern has been found where the inhibitory activity decreases when the substituent is attached at ortho or meta position. On other hand, 4d inhibitory activity is on par with 4c. It suggests that substituent (-OR and -OH) at para position play important role in the activity. In brief, some synthesized sulfonamide-substituted 1,3,5-triphenyl pyrazoline derivate have ability to prevent L-tyrosine from being oxidized. Compound 4c, 4d and 4e have better inhibitory activity compared kojic acid as reference drug and 4c had the best activity among them.

Molecular Docking
Molecular docking was conducted to predict the binding orientation of pyrazoline with tyrosinase enzyme and also to ensure the in vitro results (Jusril et al., 2020). Generally, re-docking of native ligand from the protein (i.e., tyrosinase) was used to validate the selection of docking protocol. In this study, re-docking of native ligand (tropolone) has the binding free energy of -8.1907 kcal/mol with root mean square deviation of 1.4149. RMSD is also play an important role to evaluate the docking accuracy with the value of acceptable range less than 2 (Castro et al., 2017;Makeneni et al., 2018;Yusuf et al., 2008). Furthermore, the validated docking protocols can be used for docking compound 4a-4f. Pose with the lowest binding free energy was selected as the best docked-pose and the docking results are depicted in Table 2.   Table 2, it seemed that compound 4a-4f has the binding free energy lower than the binding free energy of kojic acid. All compounds except tropolone have hydrogen bonding interactions in the active site. Notably, 4c and 4e have three hydrogen bonding with Gly281, Arg268, Glu322 and His244, Val283, Glu322, respectively. In opposite, compound 4a was only able to form hydrogen bonding interaction with Arg268. In addition, compounds 4c have highest bonding similarity with factor of binding of 12 compared to kojic acid. Factor of binding is the probability for receptor-ligand binding to the same amino acid with the positive control. These probably caused that these compounds become active. Hydrogen bonding and factor of binding may use to predict which compounds will be more active as tyrosinase inhibitor. Furthermore, root mean square deviation is also play an important role for prediction of potentially bioactive compound. Based on the docking results predict that 4c to be active compound because of some reasons. Firstly, this compound has the lowest binding free energy value of -9.1021 kcal/mol. Secondly, 4c was constructed three hydrogen bonding through amino acid residues Gly281, Arg268, Glu322 and exhibited van der Waals interaction with His61 inside the binding site of the tyrosinase. Most importantly, 4c interact with copper atom which have significant role in tyrosinase enzymatic activity. The result suggested that the existence of hydroxyl group in para position may enhance the ability to make this molecule bind into the binding pocket with purposing to maintain a high inhibitory activity. The spatial arrangement of 4c with the protein is depicted in Figure 3.
The biological assay proved 4c is the most active compound as tyrosinase enzyme inhibitor with IC50 value of 30.14 µM. Similarly, 4d and 4e compounds are active in both molecular docking study and in vitro assay but slightly weaker compared to 4c. On other hand, despite good binding free energy and binding interaction of 4a, 4b, and 4f in molecular docking, the inhibitory activity of them is very weak in experiment. This indicated that these compounds are active compounds but uncompetitive inhibitor. An uncompetitive inhibitor results inactive enzymesubstrate-inhibitor complex by binding to the enzymesubstrate complex. This is the main reason from docking results shown that these compounds were able to bind well with the protein but the opposite results from the biological assay (Lu et al., 2020)

CONCLUSIONS
In this study, a series of 1,3,5-triphenyl pyrazolines contain sulfonamide, hydroxy, and methoxy group (4a-f) have been successfully synthesized via threecomponent reaction in a sealed-vessel reactor. The chemical structures of all the synthesized compounds were confirmed by spectroscopic analyses and their tyrosinase inhibitory activity were evaluated through molecular docking and in vitro evaluation against tyrosinase enzyme. Compounds 4a, 4b, and 4f have low inhibitory activity with IC50 values > 100 µM. On the other hand, compounds 4c, 4d, and 4e surprisingly showed significant inhibitory activity to tyrosinase enzyme, stronger than kojic acid as reference inhibitor. Among all synthesized compounds, 4c exhibited the lowest IC50 value of 30.14 µM. This result revealed that the substituents at para position is preferable. The result of in vitro evaluation also agreed with the docking result. Binding mode of 4c indicated that p-sulfonamide and p-hydroxyl substituents play important role to provide hydrogen bond interactions with essential amino acid residues in the active site of the enzyme. Thus, it can be concluded that compound 4c has a very good potential for further development as a candidate for tyrosinase inhibitor.