Synthesis of Bi-Heterocyclic Sulfonamides as Tyrosinase Inhibitors: Lineweaver–Burk Plot Evaluation and Computational Ascriptions

The designed bi-heterocyclic sulfonamides were synthesized through a two-step protocol and their structures were ascertained by spectral techniques including IR, 1H NMR and 13C NMR along with CHN analysis. The in vitro inhibitory effects of these sulfonamides were evaluated against tyrosinase and kinetics mechanism was analyzed by Lineweaver– Burk plots. The binding modes of these molecules were ascribed through molecular docking studies. These synthesized bi-heterocyclic molecules were identified as potent inhibitors relative to the standard (kojic acid) and compound 5 inhibited the tyrosinase non-competitively by forming an enzyme-inhibitor complex. The inhibition constant Ki (0.09 μM) for compound 5 was calculated from Dixon plots. Computational results also displayed that all compounds possessed good binding profile against tyrosinase and interacted with core residues of target protein.


Introduction
Sulfonamides find their wide acceptance as the sulfa drugs across the world and these organic compounds have deep impacts on the biological systems owing to their numerous pharmacological activities. They are widely used to cure the bacterial infections. They find their usage as anti-cancer, anti-microbial, antiviral, anti-inflammatory and anti-tumor agents along with carbonic anhydrase inhibitors. 1 The carbonic anhydrase bustles of sulfonamides possess an appreciated treatment for Alzheimer's disease. 2 They are also used as anti-convulsant, anti-fungal and enzyme inhibitors. 3,4 The sulfonamides bearing piperazine moiety have enhanced their role as antipsychotic compounds to cure paranoid schizophrenia, mental depression, nausea and as anticonvulsants against electroshock (MES) induced seizures. 5,6 Certain other piperazine derivatives have also antimicrobial and antimalarial potentials. 7,8 Piperdine derivatives have been reported to have anticancer activity. 9 Some sulfonamide based piperidine derivatives have an efficient role as acetylcholinesterase inhibitors. Several other substituted piperidine based derivatives have the potential of inhibition of ureases and α-glucosidases. [10][11][12][13] Tyrosinase (polyphenol oxidase, PPO, E.C.1.14.18.1) which is a copper-containing metalloenzyme, catalyzes two major reactions in the biosynthesis pathway of melanin pigment: the hydroxylation and oxidation of monophenols to o-quinones (monophenolase activity) and the oxidation of o-diphenols to o-quinones (diphenolase acti-Abbasi et al.: Synthesis of Bi-Heterocyclic Sulfonamides as Tyrosinase ... vity). 14 Melanin is an important pigment which is found in the eyes, hair and skin of animals and especially it protects human skin against radiation. 15,16 However, excessive production and hyperpigmentation of melanin is the cause of dermatological disorders such as melasma, ephelides, chloasma, freckles, melanoderma and senile lentigines and can induce inflammation such as eczema, irritant and allergic eczema, contact dermatitis, which can result in critical and emotionally distressing trouble. 17 Besides the fact that enzymes and their activities are extremely necessary for life, the selective inhibition of critical enzymes is also considerably important for chemotherapeutic intervention in some diseases. Unregulated high enzyme activity results in the formation of reaction products at abnormal levels which can cause specific pathologies. Nowadays, the strategy of selective enzyme inhibition gets attention in modern pharmacy and enzymes have become interesting targets in drug therapies. 18 For this reason, many organic molecules have been synthesized as specific enzyme inhibitors and continue to be synthesized. Modeling methods to ascribe the three-dimensional conformations and interactions of organic molecules with the active sites of enzymes help researchers to design new drug molecules. 19 Therefore, the objective of the present study was to synthesize some new bi-heterocyclic sulfonamides, to explore their inhibitory potentials against tyrosinase enzyme and ascribe their binding interactions through molecular docking studies.

Results and Discussion
The aim of the present research work was to synthesize new biologically active compounds with low toxicity. Indeed, the current need is to introduce pharmacologically active drugs to help in pharmacy against the increasing enzyme inhibition.

1. Enzyme Inhibition Activity
The synthesized bi-heterocyclic sulfonamides, 5, 8, 11, 14, were screened against tyrosinase and their in vitro inhibitory activities are presented in Table 1. These molecules exhibited outstanding potentials, which are evident from their lower IC 50 (µM) values, relative to standard, kojic acid, having IC 50 value of 16.8320 ± 1.1600 µM. Although, the experimental activity is cumulative for the whole molecule, however, nevertheless, a partial structure-activity relationship (SAR) was established by analyzing the effect of varying heterocyclic parts on the inhibitory potential. It needs to be mentioned, that except this variation of heterocyclic moiety, all other parts were the same in these molecules. The general structural parts of the inspected compounds are labeled in Fig. 1.
The comparison of inhibitory potential of 5 (IC 50 = 0.0586 ± 0.0033 µM) and 8 (IC 50 = 0.4078 ± 0.0151 µM), revealed that the presence of an additional oxygen atom within the heterocyclic ring (4-morpholinyl group) in 8 resulted in a decrease of activity as compared to 5, in which a simple piperidinyl ring was present. The compound 5 was the most active compound in the series as well. It means, the presence of piperidinyl ring is a credible option for the promising activity of such compounds (Fig. 2).
In compound 11, a para-methyl group was present at the piperidinyl ring (4-methyl-1-piperidinyl), while in 14 two methyl groups were present at the meta-positions of this heterocyclic part (Fig. 3). When the inhibitory potential of these two molecules is compared, it was perceived that molecule 14 with two methyl groups in variable heterocyclic part behaved as a slightly better inhibitor. It means when the methyl groups were present in a pseudo-symmetrical manner at 3 and 5 positions (3,5-dimethyl-1-piperidinyl), the compound 14 made some better interactions with the enzyme relative to its mono-methylated analogue 11.  Moreover, on a closer look, it was also elucidated that compounds 11 and 14 both have substituted piperidinyl ring and possessed slightly lesser inhibitory potentials as compared to 5 in which an un-substituted piperidinyl ring was present. From this, it was conceivable that the presence of any methyl group in this heterocyclic ring may render some steric repulsions and thus tending to retard the interactions of the compound with the enzyme, although to a minor extent. Hence, the presence of an un-substituted piperidinyl ring was the most suited option for the excellent activity of such molecules.

Kinetic Analysis
To understand the inhibitory mechanism of these bi-heterocyclic sulfonamides on tyrosinase, kinetic study was performed. Based on our IC 50 results, the most potent compound 5 was selected to determine the inhibition type and inhibition constant. The kinetic results (Table 2)

3. Mushroom Tyrosinase Structural Assessment
Mushroom tyrosinase, a copper containing protein comprises 391 residues. The detail structure analysis of the target protein showed that it consists of 39% α-helices, 14% β-sheets and 46% coils. The Ramachandran plots and values indicate that 95.90% of protein residues are present in the favored region and 100.0% residues lie in the allowed region. The Ramachandran graph values show good accuracy of phi (φ) and psi (ψ) angles among the coordinates of receptor and most of residues are plunged in the acceptable region. The overall protein structure and Ramachandran graph is shown in Figure 5 (A, B).

4. 1. Glide Energy Evaluation of Synthesized
Compounds Molecular docking examination is the best approach to study the binding conformation of ligands within the

4. Ligand-Binding Analysis of Tyrosinase Docked Complexes
It was envisaged that hydrogen bonds and π-π interactions were observed in 8, 11 and 14 docking complexes. In 5-tyrosinase docking no hydrogen bonds were observed, however its binding conformation was quite similar with other ligands docking complexes. In 8-tyrosinase docking complex three hydrogen bonds were observed at Glu322 and His85. The benzene ring form hydrogen bond with Glu322 having bond length 2.61 Å, whereas the oxygen atoms of benzenesulfonyl part were involved in hydrogen bonding against His85 having bond distances 2.67 Å and 1.99 Å, respectively. In 11-tyrosinase docking couple of hydrogen bonds were observed between the benzene ring of ligand and tyrosinase residues His85 and Cys83 with bonds length 2.59 Å and 2.46 Å, respectively. Moreover, the com-     Moreover, computational results also explored the good binding profiles against target protein. All compounds (5, 8, 11, and 14) exhibited good docking energy values and bound within active region of the target protein. His85 was common in all docking results. The His85 is copper bonded residue which ensures that our ligands bind within the active region of the target protein. Literature data also ensured the importance of these residues in bonding with other tyrosinase inhibitors which strengthen our docking results. 22,23 The comparative results showed that compound 5 might be considered as a superb template for the designing of new inhibitors against tyrosinase.

1. General
All the chemicals, along with analytical grade solvents, were purchased from Sigma Aldrich, Alfa Aesar (Germany), or Merck through local suppliers. Pre-coated silica gel Al-plates were used for TLC with ethyl acetate and n-hexane as solvent system. Spots were detected by UV 254 . Gallenkamp apparatus was used to measure melting points in capillary tubes. Elemental analyses were performed on a Foss Heraeus CHN-O-Rapid instrument and were within ± 0.4% of the theoretical values. IR spectra (ν, cm -1 ) were recorded by KBr pellet method in the Jas-Scheme 1. Outline for the synthesis of 1-phenyl-4-[4-substituted-sulfonyl)benzyl]piperazines (5, 8, 11, 14). Reagents and conditions: (I) 10% Na 2 CO 3 , pH 9-10, stirring at room temperature for 3-4 h; (II) dimethylformamide (DMF), LiH, refluxed for 0.5 h, followed by addition of respective electrophile (one in each reaction) and then refluxing further for 4-5 h. co-320-A spectrophotometer. 1 H NMR spectra (δ, ppm) were recorded at 600 MHz ( 13 C NMR spectra, at 150 MHz) in DMSO-d 6 using Bruker Advance III 600 As-cend spectrometer using BBO probe.

General Procedure for the Synthesis of 1-Phenyl-4-[4-(substituted-sulfonyl) benzyl]piperazines (5, 8, 11, 14)
1-Phenylpiperazine (0.2 g, 4) was added in DMF (5 mL) contained in a 250 mL round bottom flask at room temperature, added one pinch of LiH and stirred for 30 min. Then the respective electrophile from 3, 7, 10, 13 (one in each reaction) was added in equimolar amount and stirred for 4-5 h. The completion of the reaction was monitored by TLC and after its completion, the reaction mixture was quenched with ice cold water (100 mL). Consequently, the respective derivatives (5, 8, 11, 14) were collected through filtration in purified form.

4. 1. Retrieval of Tyrosinase in Protein Preparation
Wizard The mushroom tyrosinase structure was retrieved from Protein Data Bank (PDB) (www.rcsb.org) with PD-BID 2Y9X 29 in protein preparation wizard. The selected protein structure of tyrosinase was pre-processed and minimized using default parameters in Maestro interface.

4. 2. Grid Generation and Molecular Docking
Prior to molecular docking, the optimized tyrosinase structure was prepared using the "Protein Preparation Wizard" workflow in Schrödinger Suite. Bond orders were assigned and hydrogen atoms were added to the protein.
The structure was then minimized to reach the converged root mean square deviation (RMSD) of 0.30 Å with the OPLS_2005 force field. The active site of the enzyme was defined from the co-crystallized ligands from Protein Data Bank and literature data. 30 Furthermore, docking experiment was performed against all synthesized ligands (5, 8, 11 and 14) sketched by 2D sketcher in Maestro and target protein by using Glide docking protocol. 31 The predicted binding energies (docking scores) and conformational positions of ligands within active region of protein were also performed using Glide experiment. Throughout the docking simulations, both partial flexibility and full flexibility around the active site residues were performed by Glide/ SP/XP and induced fit docking (IFD) approaches. 32,33

Conclusion
A structurally unique series of sulfonamides, hybrid with a piperazine, and heterocyclic secondary amines, was synthesized and recognized with very superb tyrosinase inhibition. It was postulated from the SAR studies that molecules particularly bearing un-substituted or symmetrically methylated piperidinyl moiety, generally inhibited the tyrosinase in an excellent manner. So, it was concluded that molecule 5 in particular, and all these bi-heterocyclic sulfonamides in general, can be utilized as leading medicinal scaffolds for the treatment of melanogenesis.