Tel-Cu-NPs Catalyst: Synthesis of Naphtho[2,3- g ]phthalazine Derivatives as Potential Inhibiters of Tyrosinase Enzymes and Their Investigation in Kinetic, Molecular Docking, and Cytotoxicity Studies

: Novel one-pot synthesis naphtho[2,3-g]phthalazine ( 1a – 1k ) of Mannich base derivatives can be achieved via grindstone chemistry using a Tel-Cu-NPs (telmisartan-copper nanoparticles) catalyst. This method o ﬀ ers e ﬃ cient mild reaction conditions and high yields. Tyrosinase inhibitory activity was evaluated for all synthesized compounds, along with analysis of kinetic behavior and molecular docking studies. The synthesized compound, 1c was (IC 50 = 11.5 µ M) more active than kojic acid (IC 50 = 78.0 µ M). Lineweaver Burk plots were used to analyze the kinetic behavior of the most active compound 1c , it was reversible and competitive behavior. Compound 1c and kojic acid occurred in the presence of 2-hydroxyketone, which has the same inhibitory mechanism. The molecular docking of compound 1c and the control kojic acid were docked against 2Y9X protein via the Schrodinger Suite. The compound 1c showed a respectable dock score ( − 5.6 kcal / mol) compared to kojic acid with a dock score of ( − 5.2 kcal / mol) in the 2Y9X protein. Cytotoxicity activity was also evaluated by using HepG2 (liver), MCF-7 (breast), and HeLa (cervical) cancer cell lines, and high activity for 1c (GI 50 = 0.01, 0.03, and 0.04 µ M, respectively) against all cell lines was found compared to standard and other compounds. Therefore, this study succeeded in testing a few promising molecules as potential antityrosinase agents.


Introduction
Out of several green chemistry methodologies, one simple and rapid methodology to prepare chemical and pharmaceutical compounds is via grindstone chemistry. This method was developed by Toda et al., who showed that many, but not all, reactions can be carried out by just grinding or triturating the solids together [1]. The Mannich reaction, which is a popular reaction in organic and medicinal chemistry domains [2], is a condensation reaction that forms an imminium-ion by enol-forming a carbonyl compound. The presently known Mannich type reactions face significant challenges. One of the prime challenges is the reaction time. Other challenges include harsh reaction conditions, toxicity, catalyst requirements, and the tedious methods required to separate and purify the final product(s). Some of the proposed methodologies to address these challenges include synthetic methodologies; for example,

Synthesis of Catalysis
Scheme 1 shows the route of preparation of telmisartan nanoparticles (Tel-Cu-NPs). First, the CuCl 2 ·2H 2 O was mixed with telmisartan in ethanol with grinding for up to 10 min. A light blue solid material was obtained, and this solid material was mixed with 1 M NaOH and maintained at pH = 7. The obtained solid material was converted into a sky blue precipitate, followed by addition , where D(hkl) is the size, k is the shape constant (0.89), λ is the incident X-ray (Cu kα source, λ = 0.15405 93 nm), β is the full width at half-maximum, and θ is the incident angle of the X-ray.   102 Scheme 1. Synthesis of Tel-Cu-NPs (telmisartan-copper nanoparticles).

Powder X-Ray Diffraction Studies
The structural analysis and phase crystallinity of the synthesized Cu-NPs were examined via the powder X-ray diffraction method. Figure 2 shows the Cu nanoparticle diffraction pattern at a 2θ value of 43.20 • , 50.42 • , and 74.15 • with respect to the (111), (200), and (220) planes individually. A copper cubic lattice formation can be seen. JCPDS No. 040836 indicates that there was good agreement compared with the standard pattern for a pure face centered cubic of Cu-NPs. There were no impurity peaks. From the observed main diffracted peak, the average crystalline size could be calculated using the Scherer equation: where D (hkl) is the size, k is the shape constant (0.89), λ is the incident X-ray (Cu kα source, λ = 0.15405 nm), β is the full width at half-maximum, and θ is the incident angle of the X-ray. The average crystallite size of the synthesized copper nanoparticles was 25.41 nm.  Figure 3 shows the morphology of the SEM images of the Cu-NPs obtained in water. A 200 nm particle size was found, conforming to the formation of Cu nanoparticles from telmisartan. Figure 4 shows the EDX of Cu-NPs that exhibited the elements Cu, C, and O in the nanoparticles, conformed by the formation of the nanoparticle.  Naphtho [2,3-g]phthalazine (1a-1k) was synthesized as shown in Scheme 2. First, the 1,4-dihydroxyanthraquinone of 1 with aldehyde (2.1 equiv.) in the presence of hydrazine hydrate (1.0 equiv.) and Tel-Cu-NPs catalysis in EtOH at RT gave naphtho[2,3-g]phthalazine with a 95% yield. Compounds of 1a-1k were purified by column chromatography, and their structure was determined by spectroscopic analysis. Assignment of the selected characteristic IR bands of compounds 1a-1k exposed the functional groups OH, NH, and C=O, which obtained strong absorption band ranges between 3406-3414 cm −1 , 3280-3286 cm −1 , and 1750-1754 cm −1 , respectively. The 1 H-NMR spectra show signals at δ 5.30-5.39 and 9.20-9.86 ppm corresponding to the OH and NH protons, respectively. The 13 C NMR spectrum shows signals from δ 187.1-187.6, 151.5-153.9, and 50.7-60.8 ppm, that correspond to the C=O, -C-OH, and -C-N carbon atoms individually. In addition, mass spectra show that all compounds of molecular weight conformed to molecular ion peaks present in the mass spectral analysis.  Figure 5 shows the recovery of the catalyst salvaged from at least 10 run times, with a slight loss in catalytic activity. The lessening of activity could be detected with the regenerated catalyst on salvaging due to the surface area of the catalyst during the reaction, or partial loss of the basic sites/regeneration. The application of the catalyst was inspected by optimizing the reaction conditions. A number of aldehydes were selected for the condensation reaction with the Cu-NPs (1 mole %) catalyst at room temperature in a solvent-free setting, and the yield is reported in Table 1. The Cu-catalyzed performance and reaction mechanism are shown in Scheme 3.

Inhibitory Mechanism
2-Hydroxyketone is in compound 1a-1k and kojic acid, and plays a major role in their mechanism. The compound 1c had the highest inhibition; the mechanism of inhibition is represented in Scheme 4 [26]. The kinetic behavior was studied using compound 1c at different concentrations with respect to L-DOPA, by mushroom tyrosinase. Figure 6 shows the lineweaver Burk plots, which indicated 1c is a competitive inhibitor. The result, due to the abscisse 1/[L-DOPA] is the reciprocal of the L-DOPA concentrations and reaction rate of tyrosinase activity.

Molecular Docking
The compound 1c and control Kojic acid were docked with 2Y9X protein via Schrodinger Suite. The compound 1c showed a dock score (−5.6 kcal/mol) higher than that of kojic acid, with a dock score of (−5.2 kcal/mol) in 2Y9X protein. The hydrogen bond stretches of compound 1c and kojic acid were strong hydrogen bonds, and 3.5Å in 2Y9X. The 1c forms three hydrogen bonds on the 2Y9X protein.
The amino acid residues Arg268 (bond length: 5.85), Ser282 (bond length: 4.25), and Val283 (bond length: 3.62) were intricate in the hydrogen bonding interface. Residues of His61, His85, Phe90, His94, Phe192, Trp227, Val248, Gly249, Met257, Asn260, Thr261, His263, Phe264, Leu275, Pro277, Met280, Gly281, Pro284, His285, Ala286, Phe292, and His296 were involved in the hydrophobic interactions. The connections of 1c with the 2Y9X protein are shown in Figure 7. One hydrogen bond is formed from the control, kojic acid, in the 2Y9X protein receptor. The amino acid residue interaction with hydrogen bonding was involved in His263 (bond length: 1.79). Hydrophobic interactions were involved in the amino acid residues of Met280, Val283 and His296. The 2Y9X protein interactions with the control kojic acid are shown in Figure 8. The results of compound 1c show the better inhibition than the control kojic acid in mushroom tyrosinase protein 2Y9X. The tyrosinase enzyme inhibition values are listed in Table 3.

Synthesis of Telmisartan Mediated Copper Nanoparticles (Tel-Cu-NPs)
The mixture of CuCl 2 · 2H 2 O (0.5 mmol, 5 mL), and telmisartan (1 mmol, 10 mL) was added to 10 mL of ethanol, then the reaction mixture was given 1 M NaOH with stirring and maintained at pH 7. The reaction mixture converted to a blue precipitate followed by the addition of 15 mL of water. The bile precipitate was obtained, then the precipitate was filtered and purified with suitable techniques.

General Preparation of Compounds (1a-1k)
To a solution of 1,4-dihydroxy-anthraquinone (16.02 g, 0.10 mol) in EtOH (150 mL), aldehyde (0.30 mol), hydrazine hydrate (2.44 g, 0.02 mol), and catalyst Tel-Cu-NPs (1.0 mg) were mixed in a mortar at RT. Achievement of the product as checked by TLC. The insoluble crude product was filtered, then the final product was separated from the column chromatography using suitable eluting solvent. Similarly, compounds (1b-1k) were synthesized by using the same method.
Catalyst recovery studies: In order to investigate the reusability of the catalyst, after completion of the reaction, copper nanoparticles were recovered by filtration of the mixture in a vacuum and thoroughly washed with ethylacetate. The final product was soluble in EtOAc solvent and we then separated the final product from the mixture. The copper nano catalyst was thoroughly washed with water, followed by filtering and then drying in an oven at 60 • C, and was then used for the next reaction without any reactivation [27].

Anti-Tyrosinase Activity
The mushroom tyrosinase (powder, ≥1000 unit/mg solid, EC 1.14.18.1) inhibitory activities were measured spectrophotometrically, as defined previously with some modifications [28]. For preparation of the test solution, 12.428 U of mushroom tyrosinase was added to L-DOPA 1.5 mM and 0.1 mM sodium phosphate buffer (pH 6.5), and incubated at 30 • C for 2 min. A total of 3.0 mL of test sample was where A = absorbance of incubation (after) of the blank solution; B = absorbance incubation (before) of the blank solution; C = absorbance of incubation (after) the blank solution; D = absorbance of incubation (before) the blank solution.

Grid Generation and Molecular Docking
The tyrosinase structure was arranged via "Protein Preparation Wizard" by the Maestro interface in the Schrodinger Suite. Originally, bond orders were allotted and the target protein was added. Later, the protein was minimized to attain the congregated RMSD-root mean square deviation of 0.30Å via the OPLS_2005 force field. The active position (binding pocket) of the target protein was demarcated by literature survey and the Protein Data Bank [31][32][33]. Compound 3c and control kojic acid were drawn by using 2D sketcher in the Schrodinger Suite for docking studies. The studies were achieved by the target protein with a Glide docking procedure to forecast the conformational positions and docking scores (binding energies) of the ligands inside the active area of the protein. All over the docking mock-ups, both partial and suppleness of the active site remains was achieved by Glide/SP/XP and induced fit docking (IFD) methods. The inhibitor devouring minimum binding energy value that was best scoring, and the outcomes, were examined via the Discovery Studio 2019 software package.

Cytotoxic Activity
The compounds (1a-1k) were evaluated for cytotoxic activity using the method in our literature report [34]. The details of the experiment are presented in the Supplementary Materials. The cytotoxic values were confirmed based on at least three independent evaluations.