Cu(II)-Promoted the Chemical Synthesis of New Azines-Based Naphthalene Scaffold as In Vitro Potent Mushroom Tyrosinase Inhibitors and Evaluation of Their Antiproliferative Activity

Abstract Herein, synthesis of new designated azines-based naphthalene scaffold (3a–10) was assisted by CuCl2.2H2O for the first time. Their biological screening manifested that azines 3a, 4, 7, 9, and 10 exhibit effectual mushroom tyrosinase inhibition with IC50 values within the ambit of 3.75–12.36 µM. Mostly, azines 4 and 9 demonstrated about four-fold enhancement in the activity (IC50 = 3.91 ± 0.16, 3.75 ± 0.15 µM, respectively) comparable to the kojic acid (IC50 = 16.86 ± 0.84 µM). Molecular docking of azines 4 and 9 against mushroom tyrosinase (2Y9X) proved the significant rule of isatin moiety (4) in hydrogen bonding, the importance of 4-dimethylamino styryl unit (9) in hydrophobic interactions, and the overall findings confirmed the momentousness of azine group (N–N) and naphthalene scaffold in the interactions with the histidine key residues of the tyrosinase binding pocket. Evaluation of antiproliferative activity indicated that, azine 4 was the most potent one against both MCF-7 and HCT116 with IC50 values of 4.35 ± 0.18 and 2.41 ± 0.12 µM, respectively. Whereas 9 was the most robust azine toward HepG2 with an IC50 value of 2.19 ± 0.12 µM. Exhaustively, azines 4 and 9 could be promising biomedical lead candidates. Graphical Abstract


Introduction
Azines are diazabutadiene-containing compounds, in which the two imine groups are aligned in opposite orientations via N-N linkage. 1 In the organic synthesis realm, azines are recently attracted abundant momentum owing to their significance in chemical transformations to install valuable bioactive frameworks. 2 Huang and coworkers in 2014 reported that an Rh(III)-mediated C-C bond formation via cascade C-H/N-N bond activation protocol to transform ketazines with alkynes to the corresponding isoquinolines under mild conditions. 3 The reaction proceeds at room temperature with air atmosphere as an external oxidant and the catalytic cycle was proposed through the formation of rhodacycle key intermediates that are enabled by azine permanent directing group ( P DG). By replacing the expensive Rh(III) catalyst [Cp Ã Rh(H 2 O) 3 (OTf) 2 ] with air-stable Co(III) complex [Cp Ã Co(CO)I 2 ], Bhanage et al. in 2019 disclosed the creation of isoquinolines by the same pathway with features including high atom economy, good yields, wide substrate scope, free external oxidant as well silver salt, and gram scalability. 4 In 2020, Shi and his scientific group described the first intermolecular C-N bond construction between ketazines with sulfonamides through ortho-C-H direct amidation transformation that enabled by [Cp Ã RhCl 2 ] 2 catalyst. 5 In 2021, List and Obradors reported a novel direct C-C bond formation approach between heterocyclic azines and silyl ketene acetals (SKA). 6 This straightforward C-H functionalization achieves by organosilicon-based Lewis's acid catalysis with complete C4-regioselectivity under mild conditions and high yields. Lim and Koo in 2005 reported the alkylation of aromatic aldazines by C-C bond formation with terminal alkenes via ortho-C-H bond activation route under rhodium catalytic system that composed of [RhCl(coe) 2 ] 2 and Cy 3 P. 7 Importantly as well, Tolstikov research group published their scientific article that described the synthesis of two azine-based macrolides through sequential Tishchenko and [1 þ 1]-condensation reactions. 8 Also, azines play a crucial rule in coordination chemistry domain as useful chelating agents. Transition metal (TM) complexes derived from azine ligands have been extensively studied according to their emission properties, 9,10 extraction ability, [11][12][13] and structural aspects. [14][15][16] As well, Angadi et al. reported the synthesis of ten first row TM complexes-based azine ligand and their antibacterial with antifungal activities were investigated. 17 The results indicated that the constructed TM networks exhibited weak to high antibacterial activities against Klebsiella and Pseudomonas with inhibition zone values of 6-23 mm in comparison with gentamicin (17-19 mm). The inhibition zone toward fungal strains A. Niger and A. Flavous was measured between 9 and 30 mm comparable to nistatin standard (20-22 mm).
Moreover, azines are endowed with significant physical properties and aspirational applications such as chemosensors for detection of anions, 18 cations, [19][20][21] thiols, 22 H 2 S, 23 formaldehyde, 24 and dichloromethane. 25 Besides, they are utilized to create liquid crystal displays (LCDs), 26,27 nonlinear optical materials (NLOs), 28 organic semiconductors, 29 laser dyes, 1 energetic materials, 2 and light control devices. 30 Furthermore, because of their reducing affinity, azines are excellent for use as hole-transport materials in optoelectronics. 31 Additionally, azines have been employed in the generation of image-recording materials, 1 tunable molecular devices, 32 and as building blocks to modular molecular architectures in covalent organic frameworks (COFs), 2,33-36 metal-organic frameworks (MOFs), 2,25 pollutant degradation polymers, 37,38 polymeric corrosion inhibitors, 39 and supra-molecular chemistry domain. 40 Regarding chemical synthesis of azines, several traditional protocols were reported, 1,2 in which the condensation reactions between carbonyl compounds and hydrazine or hydrazones were carried out under various conditions. In 2021, Khoshneviszadeh et al. described direct one-step synthesis of symmetrical aldazines derived from benzaldehydes and hydrazine hydrate under acid-free conditions. 41 Sankaran and Ganga in 2020, disclosed the preparation of two unsymmetrical azines of 3,5-dimethoxy-4-hydroxy benzaldehyde via two-step methodology, in which the aldimine (hydrazone) formation was promoted by Brønsted-Lowry acetic acid. 42 Said and coworkers in 2020 reported two-step construction of symmetrical and asymmetrical azines using 3,5-diphenylcyclohex-2-en-1-one as a starting material to install the corresponding hydrazone synthon that condensed with several aldehydes and ketones to access the respective aldazines and ketazines, respectively. 43 Pranowo and his group succeeded to install new symmetrical amino aldazine of 6-nitroveratraldehyde via the corresponding benzylidene-containing electron withdrawing groups or hydrazone intermediates that were treated with excess of 80% hydrazine hydrate and 10% Pd/C catalyst loading. 44 Agasimundin and Ujjinamatada proved that the Brønsted-Lowry HCl acid impacts the formation of symmetrical or unsymmetrical azines when 2-acetylbenzofuran hydrazone treated with aromatic aldehydes under acidic conditions. 45 Where, in the presence of HCl, symmetrical aldazines were produced and vice versa.
Concerning green practices for the synthesis of azines, Sankaran and Arun in 2015 demonstrated an efficient and eco-friendly approach for producing asymmetrical fluorenone azines using sulphated-titania (TiO 2 -SO 4 2À ) as acidic catalyst. 46 Monemi and his colleagues manifested onepot condensation methodology to generate asymmetrical aromatic aldazines using hydrazine sulfate and benzaldehydes under basic conditions enabled by triethylamine (Et 3 N). 47 With the same manner, this group published the synthesis of symmetrical azines through physical grinding route under solvent-free conditions. 48 Swaminathan research group achieved new successful pathway to create a wide library of benzophenone azines through greener procedures using inexpensive and recyclable BiCl 3 -K10 as a solid catalyst. 49 Most recently in 2022, Alem an et al. described in their pioneering article a practical and scalable strategy for construction of aldazines in excellent yields with 5% wt. loading of carbon nanotubes heterogeneous catalyst at room temperature under metal-free conditions. 50 Importantly, El-Hiti and coauthors in 2019 published a novel fashion to afford azines, in which phosphonic dihydrazide was used instead of hydrazine and the expected phosphorylhydrazine products were not generated. 51 Moving to TM-mediated single-step pathways for the preparation of azines, Ismail and Lasri reported that FeCl 3 -catalyzed 9-fluorenone and benzophenone hydrazones under refluxing conditions to install their symmetrical and unsymmetrical azines in excellent yields. 52 In 2020, Semeril et al. described that non-pincer-type Ru(II)-enhanced tandem protocol to furnish symmetrical aldazines in high yields utilizing primary alcohols and hydrazine hydrate as precursors under aerobic conditions. 53 In the same regard, the prior transformation was also assisted by distinct pincer type complexes such as PNP-Ru catalyst, 54 NNN-Ni, 55,56 and PNP-Mn complex. 57 On the other hand, utilizing PNP-Ru(II) pincer complex to transform secondary alcohols with hydrazine to the corresponding ketazines was only reported by Gunanathan et al. 58 To the best of our knowledge, employing CuCl 2 .2H 2 O as a catalyst for the synthesis of azines is unknown to this time, producing a workspace in the organic synthesis community. Therefore, we will be motivated to investigate it as a TM-based catalyst in our study.
Biologically speaking, azines are exhibited antimicrobial, antioxidant, and cytotoxicity with low IC 50 toward several cancer cell lines 2,42,43,59 (Figure 1). Recently, azines have been used as important lead compounds for drug designing and development due to their modulating demeanor against bio-enzymes and receptors. 2,41,60 In 2021, Piyasaengthong and coauthors documented that the thiazolylazine derivative showed inhibitory activity against pepsin and papain enzymes with IC 50 values of 29.83 ± 0.02 and 78.18 ± 0.06 mM, respectively. 60 Utmost importantly, the research group of reference 41 reported that 2,4-dihydroxybenzaldehyde azine (A) (Figure 1) demonstrated a threefold increase in tyrosinase inhibitory activity (IC 50 ¼ 7.30 ± 1.15 mM) when compared to kojic acid (IC 50 ¼ 20.24 ± 2.28 mM) as a known suppressor. As well, A showed over 82% cell viability toward A375 melanoma cell line and subsequently, could be the first reported promising azine as a tyrosinase inhibitor. Consequently herein, we hope for our work to be the second scientific article in this attractive research area. Some reported azines (A-F) with different applications are depicted in Figure 1. 2,50,53 The common structure aspects of tyrosinase active domain in different species are a dinuclear, type 3 Cu-polyphenol oxidase, in which each copper atom is chelated with three histidine (His) residues. Also, the two Cu atoms are interacted with dioxygen to access a kay intermediate, which oxidizes the phenolic substrates. Tyrosinase is found in animal and plant tissues, where located inside melanosomes, which are produced by skin melanocytes (melanin cells). During melanogenesis, tyrosinase catalyzes the oxidation of tyrosine to produce melanin via the hydroxylation of a monophenol to ortho-diphenol that oxidized to ortho-quinone, which undergoes polymerization to afford melanin pigment that serves as photoprotective and toxic metals chelator. 41 Despite the prior functions of melanin, it is uncontrolled production leading to an excessive concentration and subsequently causing challenges such as skin hyperpigmentation for humans and undesirable browning (taste and color defects) in fruits and vegetables. 41 Therefore, highly potent tyrosinase inhibitors are required for dealing with the former obstacles in medical and cosmetic sectors as well as agriculture and food production. 61,62 Another persistent challenge to humans, cancer is still one of the most intricate diseases to tackle, hence the discovery of novel antitumor agents is a vital therapeutic research topic. Among the attractive building blocks in medicinal chemistry discipline, naphthalene scaffold that able to derive a wide library of bioactive molecules, 63 particularly, in cancer treatment as promising tubulin polymerization inhibitors. 64,65 Azines-based naphthalene platform is rare in the literature. Amongst them, azine linked unsymmetrical naphthalene (F) (Figure 1), which displayed an efficient fluoride ion (F -) sensing. 66 Afriana et al. in 2021 67 have succeeded to obtain symmetrical naphthalene aldazine (E) (Figure 1) via microwave-assisted green synthesis in just nine minutes. This azine showed toxicological impact to Artemia salina Leach with LC 50 value of 47.20 mg/mL, thus it could be a potential anticancer agent. By the same token, developing of new azines coupled with naphthalene could be provide valuable bioactive molecules with numerous implementations.
Inspired by the above-mentioned literature studies, we investigated our available feedstock chemicals to assembly an appropriate design approach for the creation of novel azines-containing naphthalene moiety using CuCl 2 .2H 2 O, base-free catalytic system. After the full characterization, the installed azines have been screened for their anticancer and tyrosinase inhibitory activities.

Design approaches
To improve the biological profile of azine small-molecules toward tyrosinase biological target and encouraged by the promising tyrosinase inhibitory activity of 2,4-dihydroxybenzaldehyde azine (A) 41 ( Figure 1) and oxime based chalcone derivative (G) 62 (Figure 2) as well as the biological properties of above azines B-E (Figure 1), azine target compounds were designed using different in silico drug design strategies. 68 Starting with ring extension through fusion pathway, symmetrical ketazine 3a (as numbering in synthesis section) as lead compound was sketched. Next, applying molecular hybridization, ring variation, and chain extension tactics, azine targets 4-10 were also proposed ( Figure 2). Thence, these azines were computed to predict their physicochemical properties and druglike nature (Table 1) using ADMETlab 2.0 web platform as earlier described in 2021. 69 The computed azines have been exhibited the optimal range for each physicochemical property where, the molecular weight as an indication for the size parameter is between 100 and 600 g/mol (MW, 311.14-431.16), flexibility: no more than 11 rotatable bonds (nRot, [3][4][5], and polarity: topological polar surface area is between 0 and 140 Å 2 (TPSA, 24.72-62.10) ( Table 1). Lipophilicity is a significant physicochemical descriptor quantified by the partition coefficient logP between water and n-octanol, which displays a crucial indicator of permeability across the cell wall. 70 The predicted azines manifested logD values lower than 5 and logP slightly higher than 5 except compound 8 is out of range. Therefore, these compounds could  be having good permeability and absorption ability through the cell membrane of infected cells.
Considering the beyond informative predictions, the estimated azines passed both drug-likeness metrics Lipinski and Golden Triangle (except 8 is rejected). Therefore, they can be possible as drug lead candidates (Table 1).

Chemistry
As a new chemical synthesis approach, this research work was motivated by the previous report, 52 in which FeCl 3 -mediated the synthesis of 9-fluorenone and benzophenone azines in excellent yields. Herein, we have employed CuCl 2 .2H 2 O instead of the prior Lewis acid as a TM catalyst to enhance the construction of azines for the first time according to the best of our knowledge. The construction of scaffold substrate 2-acetylnaphthalene hydrazone (2) 66 is represented in Scheme 1.
Commercially available 2-acetylnaphthalene (1) was converted to its respective ketohydrazone 2 by condensing the hydrazine hydrate in ethanolic solution under reflux conditions. Treatment of ketimine 2 (1.0 equiv.) with catalytic amount of CuCl 2 dihydrate (0.5 equiv.) 71 in refluxing ethanol afforded the corresponding symmetrical ketazine 3a 58 in high yield (95%) after 2 h. Under the same conditions and without CuCl 2 .2H 2 O, 3a was generated as traces after 1 h, confirming the crucial rule of copper Lewis's acid as a catalyst. Also, we installed 3a in excellent yield (99%) from another pathway by condensing 2 with its respective ketone 1 under the former conditions that directly generalized as an optimal without further modification. The chemical structure of ketazine 3a was elucidated by IR, NMR, and mass spectrometry. The obtained data is matched very well with the landmark article by Gunanathan et al., 58 who also documented the single-crystal-X-ray analysis for it. In this report, 58 ketazine 3a was synthesized in 89% yield through the reaction of secondary alcohol that named 1-(naphthalen-2-yl)ethanol with hydrazine hydrate using PNP-Ru(II)/KO t Bu as a catalytic system (Scheme 1).
The catalytic cycle for the formation of 3a is like the previously reported mechanism 52 and depicted in Scheme 2. We think that this transformation is initiated by complexation between CuCl 2 .2H 2 O and hydrazone derivative 2 via coordination bond to furnish the catalytically active intermediate I that becomes more susceptible for receiving electrons (electrophilic center). Next, transit I undergoes nucleophilic addition with another molecule of 2 to provide intermediate II (hydrazonyl-hydrazine), in which the leaving hydrazine moiety becomes a weaker base and has a better leaving ability as a result for coordination with copper salt. Consequently, transit II afforded the desired product 3a after elimination of hydrazine and regeneration of CuCl 2 .2H 2 O.
Afterwards, we directed our attention toward the preparation of the corresponding unsymmetrical azines as target molecules. Catalytic condensation of hydrazone synthon 2 with various feedstock aldehydes and ketones that namely; isatin, indole-3-carboxaldehyde, 2-acetyl-3H-benzo[f]chromen-3-one, 2-(4-acetylphenyl)isoindoline-1,3-dione, fixolide, and 4-dimethylamino cinnamaldehyde under the aforementioned conditions afforded the corresponding azine products 4-9 (Scheme 3). FT-IR spectrum of 4 confirms the presence of the following functional groups; NH (3177 cm À1 ), isatinic C¼O (1734 cm À1 ), and C¼N (1604 cm À1 ). The key NMR signals of 4 proves the presence of singlet NH at d 10.89 ppm and methyl group at d 2.44 ppm and its respective carbon chemical shift is resonated at d 15.55 ppm. The mass spectrum of 4 displays the molecular ion peak at m/z¼313 (10%) that promoted its structure. The 1 H NMR spectrum of 5 exhibits a singlet signal at d 11.68 ppm that assigned to NH resonance. Moving to upfield region, methyl protons created a singlet 1 H chemical shift at d 2.51 ppm and its corresponding 13 C resonance at d 15.16 ppm. Moreover, the mid-IR spectrum of 5 signalizes the stretching vibration of indole NH around 3214 cm À1 .
The NMR spectra of 6 indicate two singlet signals at d 2.47 and 2.32 ppm, which attributable to two methyl groups and their respective 13 C chemical shift are produced at d 17.62 and 15.30 ppm, respectively. The carbonyl group that assignable to coumarin unit is resonated at d 157.93 ppm in 13 C NMR spectrum and its strong stretching vibrational mode is absorbed in the IR spectrum at 1711 cm À1 . The 13 C NMR analysis of 7 displays a 13 C resonance at d 167.38 ppm, which attributable to the carbonyl of isoindoline-1,3-dione. EI-MS stick diagram of 7 displays the heaviest molecular ion peak at m/z¼431 [M] þ (10%) and the tallest base peak at m/z¼127 (100%) that related to the naphthalene moiety (see supporting information figure S19).
The mid-infrared spectrum of 8 shows strong aliphatic C-H stretching vibrational freedom around 2960, 2920, and 2879 cm À1 due to the presence of eight methyl groups that resonated in the upfield region of 1 H NMR from d 2.47 to 0.95 ppm. The NMR data of 9 demonstrate CH 3 NCH 3 chemical shift at d 2.99 ppm and their respective 13   well with the parent molecule 9 albeit with free amino without methyl groups (see supporting information figure S27).
Surprisingly, when ketohydrazone 2 condensed with cinnamaldehyde, phenylacetaldehyde, and propionaldehyde under the above-mentioned conditions, the symmetrical ketazine 3a was obtained as a single product ( 1 H NMR) in 78%, 58%, and 12% yield, respectively (EI-MS data). After that, our efforts were focused on the use of available dialdehyde like glyoxal to furnish the target molecule of symmetrical azine 10 (Scheme 3). FT-IR spectrum of 10 showing the absorption band of C¼N with wavenumber of 1584 cm À1 , which relatively decreased than the prior compounds. 21 Also, EI-MS spectrum supported the chemical structure of 10 with molecular ion peak at m/z¼390 [M] þ (3%) and the naphthalene fragment as a base peak at m/z¼127 (100%). After a successful installation of 10, we attempted to extend this trial to include another dicarbonyl compounds that available in our feedstock such as glutaraldehyde, acetylacetone, and acetonylacetone (2,5-hexanedione), surprisingly again, we got the symmetrical ketazine 3a as pure compound in 64%, 57%, and 14% yield, respectively. Interestingly, in the case of glutaraldehyde, we noticed that the upfield region of NMR spectra contains two singlet signals at d 2.44 ( 13 C 15.17) and 2.38 ( 13 C 15.15) ppm, besides 28 1 H resonances in the aromatic region, whilst the other 3a data show only the half of these chemical shifts. Consequently, 3a could be obtained in approximately equal ratio (NMR data) with its Z-isomer 3b (Scheme 4) (see supporting information figure S33).
Collectively exhaustive, we think that the desired compounds (asymmetrical azines) were formed firstly and then attacked by another molecule of ketohydrazone 2 under these catalytic conditions to furnish while reflux the symmetrical ketazine 3a as a solid product through intermediates III and IV incorporated with the catalytic cycle that shown in Scheme 5. 52 Our interpretation for the successful formation of target azines (4-10) in some cases and others failed may be due to the different relative stabilities and reactivities of the desired compounds toward ketohydrazone 2 under these conditions. Interpretation of the prior results needs further studies that are still under investigation in our lab to achieve a better understanding. As a trial to get the desired azine, we treated 2 with cinnamaldehyde in the presence of Cu(II) salt at room temperature for two hours, but 3a was isolated again. The same thing occurred with acetic acid instead of copper catalyst. Finally, we succeeded to install the desired unsymmetrical azine 11 with high yield (88%) when the reaction was conducted under free-catalyst conditions (Scheme 6). This confirmed the prior mechanism in which the copper salt triggers the nucleophilic addition step in some cases according to the reactivity of intermediates. Importantly, 11 was isolated as cis isomer with olefinic vicinal protons at d 7.22 ppm with J value of 8.4 Hz. Table 2 shows the inhibitory activities of all the synthesized azines 3a-10 toward mushroom tyrosinase in terms of IC 50 values. For comparison, kojic acid was employed as a positive control. 41 Scheme 4. Formation of 3a and 3b (in the case of glutaraldehyde) as side products.

In vitro mushroom tyrosinase inhibitory potency
Scheme 5. Proposed mechanism for the formation of symmetrical ketazine 3a as a side product.
Four azines (3a, 4, 7, 9, and 10) in vitro inhibited tyrosinase significantly with IC 50 values ranging from 3.75 to 12.36 mM. Whilst, the other compounds are inactive at the examined concentrations with IC 50 higher than 100 mM. The obtained results indicated that the lead symmetrical ketazine 3a displayed good tyrosinase inhibitory activity with an IC 50 value of 7.29 ± 0.52 mM. Replacement of 2-naphthyl unit with isatin moiety in azine 4, approximately improved the inhibitory impact on tyrosinase by two-fold with an IC 50 value of 3.91 ± 0.16 mM. The isosteric variation of isatin with indole core in compound 5, destroy the inhibitory activity (IC 50 > 100 mM). Ring extension and variation of 2-naphthyl group with coumarin and fixolide moieties in asymmetrical azines 6 and 8, respectively, also demolish the potency. Whilst in 7 that contain isoindoline-1,3dione fragment, tyrosinase inhibitory activity was enhanced with an IC 50 value of 5.72 ± 0.35 mM. Introducing of 4-dimethylamino styryl subunit as an alternative to 2-naphthyl group in azine 9, resulting in the superior inhibitory potency against tyrosinase enzyme (IC 50 ¼ 3.75 ± 0.15 mM).
Moving to the diazine derivative 10, which demonstrated inhibitory activity with an IC 50 value of 12.36 ± 0.75 mM that closer to the well-known kojic acid inhibitor (IC 50 ¼ 16.86 ± 0.84 mM). In general, azines 3a, 4, 7, 9, and 10 outperform kojic acid, particularly 9 might be a promising candidate that needs further studies aiming to gain a valuable cosmetic agent as a tyrosinase inhibitor.

In vitro antiproliferative activity
Using the standard sulphorhodamine B (SRB) assay, 72 the antiproliferative potency of all the constructed azines 3a-10 was estimated in vitro toward three human cancer cell lines, breast cancer (MCF-7), colon carcinoma (HCT116), and liver cancer (HepG2) in comparison to doxorubicin as a reference drug. The assay was assessed at various doses ranging from 0 to 100 mM (Figure 3). The obtained findings were represented as proliferation inhibitory concentration (IC 50 ) values that are outlined in Table 3 and are an average ± SD of two separate experiments. In comparison with doxorubicin, azines 3a, 4, 9, and 10 showed significant anti-proliferative activities against the tested cancer cell lines. Whereas the other azines 5-8 exhibited moderate to weak potencies. Mostly clearly, HepG2 was the most susceptible carcinoma cell line to the impact of the new azines.
Unsymmetrical azine 4 that bearing isatin moiety is found to be the most potent one against both MCF-7 and HCT116 with IC 50 values of 4.35 ± 0.18 and 2.41 ± 0.12 mM, respectively. Whereas 9 that contains 4-dimethylamino styryl fragment, is the most robust azine toward Scheme 6. Chemical synthesis of the desired unsymmetrical azine 11.    HepG2 with an IC 50 value of 2.19 ± 0.12 mM. Importantly as well, symmetrical diazine 10 is more active than doxorubicin (IC 50 (Table 3).

Cell cycle analysis and apoptosis
Cancer cells are dividing out of control, which leads to the development of a tumor. Thus, cancer treatment relies so heavily on understanding the science behind cell proliferation and cell cycle regulation. 72 Azine 9 as the most potent antiproliferative one against HepG2 with an IC 50 value of 2.19 ± 0.12 mM, was selected to assess the percentages of cancerous HepG2 population in the different phases of the cell cycle using flow cytometric analysis. 73 Like SRB antiproliferation assay, DMSO and doxorubicin were utilized as negative and positive controls, respectively. The obtained results are depicted in Figure 4. The DMSO-treated HepG2 demonstrated a normal cell cycle distribution pattern, where about 57% of the cells were in G1 phase, 23% were in S phase, 18% in G2/M phase and 2% were in pre-G1 phase. Whilst, positive control, doxorubicin exhibited a G2/ M cell cycle arrest with population percentage of 21.5%. To our delight, azine 9 that bearing 4dimethylamino styryl unit is induced the HepG2 population arrest with 42%, resulting in twofold increase in contrast with doxorubicin. Apoptosis is a highly programmed cell death that caused by internal factors, whereas necrosis-cell death occurs because of external impacts. 72,73 Utilizing Annexin-V/PI double staining flow cytometric analysis, azine 9 manifested a moderate HepG2 apoptotic induction with a value reaches 17.55%, in comparison to doxorubicin (31.28%) ( Figure 5). This truly proves the importance of 9 as HepG2 antiproliferative and apoptotic agents.

In silico molecular docking simulation
Following that, a molecular docking simulation was carried out to gain insight of the interactions and binding mechanism of potent azines 4 and 9 in the mushroom tyrosinase active pocket (PDB ID: 2Y9X). Molecular docking was performed by iGEMDOCK program version 2.1 74,75 and the results are summarized in Table 4. Tyrosinase catalytic site contains type-3 two copper atoms, each one is chelated with three His residues. One among them is coordinated with His61, His85, and His94, whilst the second Cu is complexed with His259, His263, and His296 ( Figure 6). 76 Literature reports proved that interactions with prior key residues can incapacitate the tyrosinase biological activity. 41,76 As manifested in Figure 7a-c, azine 4 stabilized in the active domain with fitness value of À87.334 kcal/mol through van der Waals, hydrophobic interactions, and two conventional H-bonds. Amongst them one between isatin NH and carbonyl oxygen of His85 (3.13 Å), whereas the other is installed between azine N and NH of His244 (2.91 Å), confirming the significance of azine functional group (N-N) and isatin moiety in the inhibitory activity against tyrosinase enzyme. Also, within the binding site, the electron rich naphthalene scaffold is well-fitted by several van der Waals and hydrophobic interactions such as p-p stacking with His263 and Ser282 besides p-r stacking with Val283. Asymmetrical azine 9 that exhibited tyrosinase inhibitory potency (IC50 ¼ 3.75 ± 0.15 mM) slightly better than compound 4 (IC50 ¼ 3.91 ± 0.16 mM), is overlapped into the active center of 2Y9X with lower binding energy (-98.498 kcal/mol) and the same types of interactions ( Fig.  8a-c). Where, the best docking pose shows one conventional H-bond between azine group (N-N) and Val283 (3.39 Å), while the naphthalene moiety is interacted with prior residue via p-r hydrophobic interaction (3.85 Å) and p-p stacking with His263 like azine 4. Importantly as well, 4-dimethylamino styryl unit is stabilized inside the active pocket through p-alkyl stacking with Pro277. Exhaustively, in silico docking prediction results are well-matched with in vitro tyrosinase inhibitory activity and indicated that the azine group (N-N) is responsible for hydrogen bonding, whereas naphthalene scaffold plays a crucial command in van der Waals and hydrophobic interactions (Table 4).      (Bruker, USA) at room temperature. Tetramethylsilane (TMS) is used for internal calibration ( 1 H NMR and 13 C NMR: 0.00 ppm). Chemical shifts were reported in parts per million (ppm) on the d scale and relative to residual solvent peaks (DMSO-d 6 : 1 H: 2.50 ppm, 13 C: 39.5 ppm). Coupling constants (J) are reported in Hz with the following abbreviations used to indicate splitting: s¼singlet, d¼doublet, t¼triplet, q¼quartet, m¼multiplet, br¼broad signal. Electron ionization mass spectrometry (EI-MS) spectra were attained through a GC MS-QP 2010 plus mass spectrometer (Shimadzu, Kyoto, Japan) at electron voltage 70 eV, 10 min, scan speed 1000, and within m/z range of 50-500. For chemical synthesis, starting materials and reagents were obtained from commercial sources particularly Acros Organics TM (Geel, Belgium) and used without further purification, unless otherwise indicated. Solvents were obtained from Thermo Fisher Scientific TM and employed without additional purification. All reactions were implemented in oven-dried glassware. The purity of the synthesized compounds was investigated by TLC, performed on Merck precoated silica gel 60 F 254 aluminum sheets with solvent mixture of DCM-MeOH (99-1) as eluent system. For biological screening, Agaricus bisporus mushroom tyrosinase (Enzyme Commission number: 1.14.18.1), L-3,4-dihydroxyphenylalanine (L-DOPA), kojic acid, and cytotoxicity required reagents were obtained from Merck KGaA (Frankfurter Str. 250, 64293 Darmstadt, Germany). Spots were visualized under UV illumination at 254 nm. Chemical synthesis procedures were not optimized. Compounds names are derived from ChemOffice-v19.0 and are not necessarily identical with the IUPAC nomenclature. Schemes were produced by the same version of ChemOffice. IR data were plotted by OriginPro-v2021. NMR spectra were delineated via MestReNova-v12.0.2.
Procedure  (60). The spectral data for this compound were identical to those reported in the literature. 58 See supporting information, figures S1-S4.

Mushroom tyrosinase inhibitory bioassay
Tyrosinase inhibition bioassay was accomplished through the developed method that earlier reported. 41 Where, L-DOPA was used as a substrate, kojic acid as a standard reference, and DMSO as a negative control. All the constructed azines and kojic acid were dissolved in DMSO at 40 mM as stock solutions and then diluted with phosphate-buffered solution (pH ¼ 6.8) to the desired concentrations (8 and 16 mM). The reaction was implemented by mixing 160 mL of phosphate-buffered solution (50 mM), 10 mL of requested azine/kojic acid concentration and 10 mL of mushroom tyrosinase solution (0.5 mg/mL) then added into 96-well microplates. After pre-incubation at 25 C for 10 min, 20 mL of L-DOPA solution (0.5 mM) was added into each well to a final volume of 200 mL and re-incubated at 25 C for another 10 min. Next, dopachrome development was monitored against blank by UV-Vis Spectrophotometer (Varian Cary 50 Bio) at 475 nm for 10 min. The inhibition (%) was calculated using the following equation: Inhibition (%) ¼ 100 Â (Abs control -Abs compound )/Abs control. 41 Each measurement was performed two times. The inhibitory potency of the tested azines was indicated as the concentration that inhibited 50% of the tyrosinase activity (IC 50 ).

Sulforhodamine B (SRB) colorimetric cellular-based bioassay
The sulforhodamine B (SRB) bioassay as an efficient and inexpensive methodology was utilized for in vitro screening the cytotoxicity of our azines 3a-10 toward three human cancer cell lines including breast cancer (MCF-7), colon carcinoma (HCT116), and liver cancer (HepG2) in comparison to doxorubicin as a reference drug. This bioassay was implemented according to the developed protocol by Vichai and Kirtikara. 76 The prior cancer cells were obtained frozen in liquid nitrogen (À180 C) from the American Type Culture Collection (ATCC, Rockville, MD). Prior to carrying out the assay, carcinoma cells were cultured as monolayer in RPMI-1640 medium (Gibco Glasgow, UK) that supplemented with 10% inactivated fetal calf serum (FCS, Sigma-Aldrich) and 50 mg/mL gentamycin. The cells were sub-cultured 2-3 times a week (National Cancer Institute, Cairo, Egypt) at 37 C in a humidified atmosphere with 5% CO 2 . 72 On the day before the test, cell inoculation was done, where the adherent cells were detached by addition of trypsin/ethylenediaminetetraacetic acid (EDTA) solution (0.05%/0.02% in phosphate buffered saline solution PBS; PAA) and plated in sterile 96-well microtiter culture plates in a density of 10,000 cells in 100 mL medium per well. 75 Different concentrations of azines (3a-10) were prepared from stock solutions in DMSO after dilution with culture medium (10% FCS). Each concentration (0, 6.25, 12.5, 25, 50, and 100 mg/mL) was tested in duplicate. The control contained the same concentration of DMSO as the first dilution. After incubation for 48 h at 37 C in a humidified incubator with 5% CO 2 , cells were fixed by adding 100 mL of trichloroacetic acid solution (10% wt/vol) and then incubated at 4 C for 1 h. 77 Next, plates were washed, air dried at room temperature (25 C), and each one was stained with 100 mL of SRB solution (0.057% wt/vol). 77 After that, the plates were left at room temperature for 30 min and then the excess (unbound) dye was removed by 1% acetic acid and dried at room temperature. Color intensity was measured in an ELISA reader. The relation between surviving fraction and drug concentration was plotted and IC 50 (the concentration required for 50% inhibition of cell viability) was calculated for each azine by SigmaPlot scientific graphing software. 72

Cell cycle analysis and apoptosis
Cell cycle distribution was evaluated, employing the Propidium Iodide (PI) Flow Cytometry Kit (ab139418, Abcam) followed by flow cytometry analysis. 72,73 Cell apoptosis was assessed by Annexin V-FITC/PI double staining apoptosis detection kit (K101, BioVision) utilizing a flow cytometer. 72,73 3.4. In silico computational predictions

Physicochemical descriptors and drug-likeness nature
The physicochemical properties of azines (3a-10) were estimated using ADMETlab 2.0 web platform as earlier described in 2021 (Table 1). 69

Molecular docking simulation
In silico flexible ligand docking study was exercised by iGEMDOCK program version 2.1 (Department of Biological Science and Technology & Institute of Bioinformatics, National Chiao Tung University, Taiwan), 74 which utilizes a generic evolutionary approach (GA) and an empirical scoring function to explore the interaction modes between the hit azines (4 and 9) and Agaricus bisporus mushroom tyrosinase active pocket (PDB ID: 2Y9X) as a biological target. Firstly, the three-dimensional (3 D) structures of tyrosinase was downloaded from RCSB Protein Data Bank (https://www.rcsb.org) and the co-crystallized ligand, was extracted and docked back to the corresponding binding site (RMSD 2 Å), to define the ability of docking protocol to reproduce the docking mode of the inhibitor observed in the crystal structure (iGEMDOCK validation). 75 Secondly, the two-dimensional (2 D) structure of hit azines (4 and 9) were drawn by ChemBioDraw Ultra 14.0 (PerkinElmer Informatics, Waltham, MA, USA) and converted to 3 D structure by ChemBio3D Ultra 14.0 then saved as mol format after energy minimized and Molecular Dynamic (MD) performed using MMFF94 (Merck molecular force field) method. 78 Finally, the docking process was done through drug screening (default setting) by uploading the protein pdb and ligand mol files to the iGEMDOCK program and the results were analyzed with the Discovery Studio Visualizer Client 2020 (BIOVIA, San Diego, CA, USA). Fitness value (Table  4) is the total energy of a predicted pose in the binding site. The empirical scoring function of iGEMDOCK was calculated as: 74 Fitness¼vdW þ Hbond þ Elec; where, the vdW term is pointed out to van der Waal energy. H-bond and Elec terms are denoted hydrogen bonding energy and electro statistic energy, respectively.

Conclusions
In summary, new azines-based naphthalene scaffold (3a-10) were designated and their chemical synthesis was mediated by CuCl 2 .2H 2 O for the first time according to the best of our knowledge. The installed azines were fully characterized by IR, NMR, and mass spectrometry. Next, the successful constructed azines (3a-10) were evaluated for their inhibitory potency against mushroom tyrosinase comparable to kojic acid as a positive control. Also, their antiproliferative activities and cell cycle analysis were in vitro screened toward MCF-7, HCT116, and HepG2 cancer cell lines in comparison to doxorubicin as a standard drug. Our findings from tyrosinase inhibitory activity showed that azines 3a, 4, 7, 9, and 10 outperform kojic acid (IC 50 ¼ 16.86 ± 0.84 mM), particularly 4 and 9 (IC 50 ¼ 3.91 ± 0.16, 3.75 ± 015 mM, respectively) can be promising candidates that need additional studies aiming to gain valuable cosmetic agents as tyrosinase inhibitors. Prior results are in silico confirmed by molecular docking simulation that displayed good interactions between the highest bioactive azines (4 and 9) and the pivotal residues of the tyrosinase catalytic domain. Importantly as well, azine 4 was the most potent one against both MCF-7 and HCT116 with IC 50 values of 4.35 ± 0.18 and 2.41 ± 0.12 mM, respectively. Whereas 9 was the most robust azine toward HepG2 with an IC 50 value of 2.19 ± 0.12 mM, population arrest of 42%, and apoptotic induction of 17.55%. Consequently, the promising biological outcomes and the effective installation of azines via earth-abundant Cu(II) catalyst will encourage us for further studies in these attractive research points.