tris‐Schiff Base Derivatives Containing Flurbiprofen as Potential Tyrosinase Inhibitors: Synthesis, In Vitro Evaluation, Molecular Docking, DFT and Dynamics Simulations

ABSTRACT

A novel series of tris‐Schiff bases of flurbiprofen has been synthesised via multi‐step reactions, structures were deduced by means of HR‐ESI‐MS, ¹³C‐ and ¹H‐NMR spectroscopy and screened for their in vitro tyrosinase inhibitory potential. All the synthetic derivatives attributed noteworthy inhibition except compounds (11, 14 and 16), while eight compounds (5, 6, 7, 8, 19, 24, 22 and 18) were found potent comparing them with the standard kojic acid. Furthermore, the remaining nine compounds showed good to less inhibitory activity. A para‐nitro containing compound 5 attributed the powerful tyrosinase inhibition through docking and normal mode analysis (NMA). It formed stable electrostatic interactions and hydrogen bonds that decline catalytic flexibility, as well as stabilising the enzyme in an inactive site. NMA showed disrupted strong intra‐domain coupling and global motions, signifying a broad stabilisation of the enzyme structure. Compared to kojic acid, compound 5 displayed enhanced binding stability, lower B‐factors and tighter packing. A robust correlation (R ² = 0.8754) among the IC₅₀ values and binding energy authenticated the computational model and its potential for rational inhibitor design.

New tris‐Schiff base derivatives of marketed accessible flurbiprofen were synthesised in better yields through multi‐step reactions. Structures of these compounds were deduced through ¹³C‐NMR and ¹H‐NMR spectroscopic methods and finally screened for their in vitro tyrosinase inhibitory potential.

1. Introduction

A main cellular constituent frequently detected in animals, plants, fungi and bacteria responsible for skin colour is known as melanin [1]. It exists as a complex, heterogeneous biopolymer with polyphenol‐type structure, secreted by means of melanocytes in the basal layer of dermis and exhibiting colours ranging from yellow to black [2]. Normal melanin pigmentation can protect them from ultraviolet radiation, affect vitamin‐D3 production and stop photo carcinogenesis [3]. Furthermore, many serious aesthetic issues occur from aberrant pigmentation, including senile lentigines, freckles, melasma and other types of melanin hyperpigmentation [4]. The production of melanin is mainly assisted by tyrosinase‐catalysed oxidative reactions of tyrosine [5].

A decisive enzyme in the biosynthesis of melanin in fungi, plants, bacteria and animals is tyrosinase (EC 1.14.18.1) [6]. It is an enzyme having two functions, which contains copper and catalyses the production of melanin through converting oxidising ortho diphenols to ortho quinones and monophenols to ortho diphenols. Though melanin is an important protection for the skin from UV rays, many of its by‐products result in skin cancer, freckles, melasma and age spots [7]. The inhibition of tyrosinase contributes to both the neurodegeneration linked to Parkinson's disease and the reduction of skin hyperpigmentation [8]. In the brain, tyrosinase oxidises dopamine to create melanin; Parkinson's disease is caused by dopaminergic neuron insufficiency, which is triggered by dopaquinones and results in brain damage [9]. Therefore, tyrosinase contributes to the aetiology of Parkinson's disease [10]. Tyrosinase, on the other hand, can cause vegetables and fruits to undergo undesirable enzymatic browning, which lowers their nutritional and commercial worth in the food section [11]. The most crucial tyrosinase enzyme inhibitors are hydroquinone, arbutin and kojic acid, with diverse side effects including permanent depigmentation, contact dermatitis and erythema [12]. Tyrosinase inhibitors have gathered noteworthy attention in the pharmaceutical and cosmetic sectors for their potential to avert pigmentation problems and ageing skin [13].

Within the broad category of non‐steroidal anti‐inflammatory drugs (NSAIDs), the ones with a free carboxylic acid group are extensively used throughout the world to treat a range of painful ailments, such as surgical pain, menstrual cramps and arthritis [14]. These drugs work by reducing the activity of the cyclooxygenase enzymes COX‐I and COX‐II, which reduces the synthesis of prostaglandins that are involved in inflammatory reactions [15]. Notably, NSAID use has also been connected to a lower incidence of cancer across several malignancies [16]. However, because of their strong selectivity for COX‐2 and consequently harmful cardiac effects, several COX‐2 inhibitors, such as rofecoxib and valdecoxib, have been taken off the market due to concerns about the cardiovascular risks associated with them [17, 18].

A prominent member of the non‐steroidal anti‐inflammatory drugs (NSAID) family is flurbiprofen, which has been identified as a chiral molecule and demonstrates stereoselective capabilities [19]. Flurbiprofen was first made available on the European market in 1977, and since then, it has been widely used to treat arthritis [20]. Its therapeutic range encompasses a variety of disorders, such as rheumatoid arthritis, osteoarthritis, bursitis, tendinitis, migraine, acute gout and post‐operative ocular inflammation [21]. Furthermore, vernal keratoconjunctivitis, herpetic stromal keratitis, excimer laser photorefractive keratectomy and ocular gingivitis have all been successfully treated with flurbiprofen [20, 22]. Notably, studies have shown that it may be used to control cancer in several ways, including radioprotection, post‐irradiation myeloid suppression protection and reduction of colon tumour growth in animal models [23]. However, the advent of common use‐related adverse effects, such as gastrointestinal disorders, stomach ulcers, sleepiness, allergic responses and dizziness, has undermined the efficacy of flurbiprofen and other NSAIDs and analgesic medications. Therefore, finding new drugs that are both more effective and less likely to cause side effects is crucial [24, 25].

Our group has recently reported numerous flurbiprofen derivatives and screened them for their diverse biological activities, including anti‐inflammatory [16], analgesic [26], α‐glucosidase [27], while other medicinal chemists have reported various derivatives of many NSAIDs and found them as potent biologically active agents. For example, Khan et al. reported the synthesis, urease, α‐glucosidase and α‐amylase inhibition of flurbiprofen [15, 17, 28] and albendazole derivatives with potential results [28]. Similar to this, Vladimir and his colleagues reported the anti‐inflammatory activity of naproxen derivatives with potent results [29]. Moreover, the anti‐microbial activity of ibuprofen derivatives has been reported with excellent results by Abdulla [30]. Due to the promising biological activities of commercial drug derivatives, we have synthesised some novel tris‐Schiff base derivatives of the commercially accessible flurbiprofen drug and screened them for their in vitro tyrosinase inhibitory potential. To the best of our knowledge and literature survey, this is the first report on tris‐Schiff bases based on commercially available drugs.

2. Results and Discussions

2.1. Chemistry

We have effectively synthesised twenty novel tris‐Schiff base derivatives (5–24) via multistep reactions. Initially, the marketed available flurbiprofen (1) was first refluxed with hydrazine hydrate in the existence of a coupling reagent 1,1‐carbonyldi imidazole in THF to get the hydrazide (2), which was further refluxed with diethyl 2,2'‐((4‐formyl‐1,3‐phenylene)bis(oxy))diacetate in ethanol having catalytic quantity of acetic acid to obtain esterified Schiff base compound (3). Hydrazine hydrate in excess was refluxed with the obtained Schiff base (3) in ethanol solvent to get bis‐hydrazide (4), which was refluxed with various aldehydes (aliphatic and aromatic) in the presence of acetic acid in ethanol to get the tris‐Schiff base compounds (5–24) in significant yields (Scheme 1). The thin‐layer chromatographic (TLC) method was employed to determine the reaction progress. However, the product compounds have been recrystallised with absolute ethanol to get them in pure form. Structures of these derivatives were confirmed by means of HR‐ESI‐MS, ¹H‐NMR and ¹³C‐NMR spectroscopic methods.

SCHEME 1.

Synthesis of marketed flurbiprofen tris‐Schiff base derivatives.

2.2. In Vitro Tyrosinase Inhibitory Activity

The produced derivatives were screened for their tyrosinase inhibitory activity (in vitro). All the compounds displayed varying degrees of inhibitory action except compounds 11, 14 and 16, which were found inactive. Amongst the library, eight compounds, including 5 (IC₅₀ = 8.7 ± 0.4 µM), 6 (10.6 ± 0.9 µM), 7 (13.5 ± 0.5 µM), 8 (14.0 ± 0.3 µM), 19 (14.2 ± 0.6 µM), 24 (14.9 ± 0.6 µM), 22 (IC₅₀ = 16.2 ± 0.7 µM) and 18 (18.9 ± 0.5 µM) displayed excellent inhibitory activity. Similarly, six compounds 15, 20, 21, 17, 10 and 9 showed significant activity with IC₅₀ values of 21.4 ± 0.5, 22.3 ± 0.9, 23.1 ± 0.5, 23.6 ± 0.8, 23.8 ± 0.7 and 24.6 ± 0.6, while three compounds 23, 12 and 13 attributed moderate activity having IC₅₀ values from 31.5 ± 0.6 µM to 42.6 ± 0.9 µM respectively (Table 1).

TABLE 1.

Various substituents with their IC₅₀ values.

C. no.	R	IC50 ± SEM (µM)	C. no	R	IC50 ± SEM (µM)
5		8.7 ± 0.4	15		21.4 ± 0.5
6		10.6 ± 0.9	16		Inactive
7		13.5 ± 0.5	17		23.6 ± 0.8
8		14.0 ± 0.3	18		18.9 ± 0.5
9		24.6 ± 0.6	19		14.2 ± 0.6
10		23.8 ± 0.7	20		22.3 ± 0.9
11		Inactive	21		23.1 ± 0.5
12		40.6 ± 0.3	22		16.2 ± 0.7
13		42.6 ± 0.9	23		31.5 ± 0.6
14		Inactive	24		14.9 ± 0.6

Note: Standard kojic acid = 18.7 ± 0.5 µM.

2.3. Structure Activity Relationship

To understand the effects of the synthesised derivatives on in vitro tyrosinase inhibitory activity, the compounds were divided into two groups based on attached substituents, either electron‐donating or withdrawing. A summary of structure–activity relationship (SAR) is presented in Figure 1.

FIGURE 1.

General representation of the compounds with various biological activities.

In the synthesised library, compounds with electron‐withdrawing nitro groups were found as the most prominent tyrosinase inhibitors. Compound 5 (IC₅₀ = 8.7 ± 0.4 µM), with a nitro group at the para position, exhibited excellent inhibition than 6 (IC₅₀ = 10.6 ± 0.9 µM) and 7 (13.5 ± 0.5 µM), having the same nitro groups but on different positions of the aromatic ring. Furthermore, compounds 8 (IC₅₀ = 14.0 ± 0.3 µM) and 19 (14.2 ± 0.6 µM) both the compounds have the same chlorine substituent and exhibit almost the same inhibitory activity. Similarly, comparing compounds with electron‐donating groups, including 10 (IC₅₀ = 23.8 ± 0.7 µM) with 9 (24.6 ± 0.6 µM), having the same groups at different positions, compound 10, with a methoxy group at the meta position, showed better activity than 9, having a methoxy group at the ortho position. Comparing compound 12 (IC₅₀, 40.6 ± 0.3 µM) with 13 (42.6 ± 0.9 µM), both compounds showed less inhibitory activity; the activity of these compounds might be due to the attachment of electron‐donating hydroxyl and methoxy groups to the benzene ring. In the case of aliphatic substituents, compound 22 (IC₅₀ = 16.2 ± 0.7 µM), having a butane moiety, exhibited a better inhibitory effect than 23 (IC₅₀, 31.5 ± 0.6 µM), having an n‐hexane group. We concluded that the compounds having electron‐withdrawing substituents showed excellent inhibitory effect than those of electron‐donating substituents.

The inhibitory potency of our lead compound 5 (IC₅₀ = 8.7 µM) is notably superior to the standard inhibitor, kojic acid (IC₅₀ = 18.7 µM), and compares favourably with a range of recently developed synthetic tyrosinase inhibitors. For instance, comprehensive reviews of tyrosinase inhibitors highlight that potent synthetic candidates often exhibit IC₅₀ values in the low micromolar to sub‐micromolar range [8, 31]. The high potency of compound 5 can be rationalised by a synergistic combination of key molecular properties informed by lipophilicity. Similarly, hydroxy‐based xanthone derivatives reported potent analogues with IC₅₀ values around 9.8 µM [32]. The low micromolar potency of compound 5 places it firmly among the most potent synthetic inhibitors discovered in recent years. The inherent lipophilicity of the flurbiprofen core (clogP ∼3.5 for the core structure) enhances membrane permeability and facilitates optimal partitioning into the hydrophobic active site cavity of tyrosinase, a key factor for effective inhibitor binding [33]. Our molecular docking results support this, showing the biphenyl core engaged in extensive van der Waals contacts and π–π stacking with residues like His263 and Phe264. Another factor for high potency can be rationalised by steric effects for substitution: The tris‐Schiff base architecture provides optimal steric bulk to occupy a substantial volume of the enzyme's active site. The clear superiority of the para‐substituted compound 5 over its ortho (7) and meta (6) analogues highlights the critical role of steric compatibility, a well‐established structure–activity relationship in drug design where para substitution often affords optimal binding by minimising steric clash [34]. The para‐nitro group projects into a polar sub‐pocket, enabling optimal hydrogen bonding, whereas ortho substitution introduces torsional strain that disrupts this ideal geometry. The fully conjugated system spanning the three hydrazone linkages and aromatic rings enforces molecular planarity and rigidity. This extended π‐conjugation is known to enhance binding affinity by facilitating charge‐transfer interactions and improving complementary surface contact with the active site, a distinct advantage over smaller, less‐conjugated inhibitors like kojic acid [35].

2.4. Analysis of Binding Interactions and Affinity: Insights From Molecular Docking Against PDB:2Y9X

Identifying potent inhibitors is of significant pharmacological and industrial interest. Kojic acid, a natural heterocyclic inhibitor, serves as a common reference compound but suffers from limitations like moderate potency and stability concerns. This analysis delves into the molecular interaction mechanisms of a series of novel tris‐Schiff base derivatives (5, 6, 7, 8, 18, 19, 22 and 24) with Agaricus bisporus tyrosinase (PDB ID: 2Y9X [36]) using molecular docking, elucidating structure–activity relationships based on substituent effects and comparing their performance to kojic acid. The docking results, including binding energy (BE), hydrogen bond energy (HBE) and interaction energy (E_Int), are summarised in Figure 2, with the corresponding 3D interaction diagrams presented in the accompanying Figure 3.

FIGURE 2.

Landscape for different docking scores.

FIGURE 3.

3D‐binding interaction for compounds (5, 6, 7, 8, 18, 19, 22 and 24) against 2Y9X.

The docking data (Figure 2) revealed significant variation in the binding affinity and interaction profiles of the investigated tris‐Schiff bases, strongly influenced by the nature and position of the substituents (R) on the terminal benzylidene rings. Compound 5 (4‐NO₂) exhibits the most potent inhibitory activity (IC₅₀ = 8.7 ± 0.4 µM) and the highest docking affinity (BE = −6.660 kcal/mol). This correlates with a favourable E_Int (−3.987 kcal/mol). The symmetric para‐nitro groups are strategically positioned to engage in strong electrostatic interactions and potential H‐bonding with polar residues (e.g., Arg164, Glu180, Thr301 and Asp295) lining the entrance to the binuclear copper centre.

The electron‐withdrawing nature of the nitro group enhances the polarity of the synthetic derivatives, potentially strengthening coordination or H‐bonding with key residues near the coppers (Thr306). The moderate E_H.B (−0.791 kcal/mol) suggests H‐bonding is present but not the sole driving force; hydrophobic contacts involving the fluoro‐biphenyl core and the phenyl rings likely contribute significantly. The binding mode of the most active compound 5 provides a structural basis for its high potency (Figure 3). This ligand is effectively anchored within the active site through an extensive network of hydrogen bonds. Specifically, the hydrazide moieties form critical H‐bonds with the side chains of Arg164, Glu180, Thr301 and Asp295. Furthermore, an ether oxygen atom within the ligand's core structure establishes an additional H‐bond with Thr306. This multi‐point interaction network securely positions the inhibitor, likely obstructing substrate access or stabilising an inactive conformation of the enzyme, which accounts for its low IC₅₀ value.

Two isomerase (6 (3‐NO₂) and 7 (2‐NO₂)) exhibited reduced activity (IC₅₀ = 10.6 and 13.5 µM) and slightly lower BE (−6.523 and −6.553 kcal/mol) compared to 5. The meta‐nitro (6) exhibits an exceptionally strong E_H.B (−10.719 kcal/mol), indicating formation of very strong or multiple H‐bonds. However, its E_Int (−4.903 kcal/mol) is less favourable than 5, suggesting suboptimal hydrophobic packing or steric clashes negating some H‐bond gains. The ortho‐nitro (7) has the lowest RMSD (1.312 Å), implying a very stable pose prediction, with a good E_H.B (−5.147 kcal/mol) and the most favourable E_Int (−6.658 kcal/mol) among all compounds. Its lower IC₅₀ compared to 6 suggests the ortho position, while potentially sterically demanding, allows favourable interactions that compensate for the electronic effect being less optimal than para. The influence of the substituent's position on the benzylidene ring is clearly demonstrated by comparing the nitro‐isomers 5 (para), 6 (meta) and 7 (ortho). While all three exhibit strong binding affinities, the para‐substituted compound 5 is the most active. The docking pose of compound 6 determined a significant reorientation within the pocket, engaging with a different set of residues, including Pro172, Asp173 and Thr176. Similarly, the ortho‐nitro group in compound 7 induces another distinct binding conformation, with interactions noted with Gln178, Thr176 and Asn181. This positional variance highlights that the para substitution in compound 5 allows for the most optimal geometry to engage with the key residue constellation (Arg164, Glu180 and Asp295), leading to superior inhibition. The nature of the substituent also plays a crucial role. The para‐bromo derivative 24 (BE = −6.482 kcal/mol) adopts a binding conformation analogous to that of compound 5, forming critical hydrogen bonds (E_H.B = −7.631 kcal/mol) with Arg164 and Asp295, which is consistent with its high potency (BE = −6.482 vs. −5.913 kcal/mol). In contrast, the ortho‐chloro compound 19 (BE = −6.117 kcal/mol) shows interactions with Arg164, Glu180 and Thr176, reflecting a slightly different but still effective binding mode. The hydroxy‐substituted analogue 18, displaying the weakest inhibitor (IC₅₀ = 18.9 µM; BE = −5.798 kcal/mol), also leverages its polar groups to form hydrogen bonds (E_H.B = −9.865 kcal/mol) with key active site residues like Arg164, Glu180 and Asp295, affirming the importance of these interactions for inhibition. The slightly lower potency of these compounds compared to 5 may be attributed to subtle differences in the geometry and strength of these hydrogen bonds. The aliphatic butylidene 22 derivative, moderate activity (IC₅₀ = 16.2 µM) via hydrophobic filling (E_Int = −3.919 kcal/mol), while lacking an π‐stacking capability through the presence aromatic ring at this position, still achieves a favourable binding energy (−6.149 kcal/mol) through interactions with Arg164, Gln178 and Asn304, demonstrating the versatility of the core scaffold. Generally, SAR displayed substituent effects on activity and affinity. The compounds with EWGs were arranged as nitro > halogen. Para‐NO₂ (5), which maximises potency by polarising hydrazone ─NH for Cu‐coordination/H‐bonding, stabilising binding via electrostatic interactions. Activity order: para (5) > meta (6) > ortho (7). Para aligns optimally with polar residues; ortho induces a steric penalty but enhances hydrophobic contact. Compounds with halogens arranged: 4‐Br (24) > 2‐Cl (19) > 4‐Cl (8). Bromine's polarizability enhances halogen bonding. Ortho positioning improves hydrophobic complementarity. EDGs at 3‐OH (18) reduce affinity by 10‐fold versus 5. EDGs diminish hydrazone electrophilicity, critical for copper chelation. Aliphatic group at butylidene (22) shows higher BE than 18 (−6.149 vs. −5.798 kcal/mol) but less IC₅₀ than 5, confirming aromatic EWGs optimise interactions. Kojic acid exhibits weaker inhibition (IC₅₀ = 18.7 µM; BE = −4.541 kcal/mol) than tris‐Schiff base compounds (Table 2).

TABLE 2.

Docking energy and biological activity data, including calculated pIC₅₀ values, for the most active inhibitors against tyrosinase (PDB: 2Y9X).

C. no.	IC50 ± SEM (µM)	pIC50	BE (kcal/mol)	rmsd	E_H.B	E_Int
5	8.7 ± 0.4	5.061	−6.660	1.938	−0.791	−3.987
6	10.6 ± 0.9	4.975	−6.523	1.513	−10.719	−4.903
7	13.5 ± 0.5	4.870	−6.553	1.312	−5.147	−6.658
8	14.0 ± 0.3	4.854	−5.913	1.107	−2.235	−3.990
18	18.9 ± 0.5	4.724	−5.798	1.777	−9.865	−4.833
19	14.2 ± 0.6	4.848	−6.117	1.168	−4.783	−4.655
22	16.2 ± 0.7	4.791	−6.149	1.364	−3.838	−3.919
24	14.9 ± 0.6	4.827	−6.482	1.163	−7.631	−4.315
Kojic acid	18.7 ± 0.5	4.728	−4.541	1.151	−7.320	−10.500

To further validate the predictive power of the molecular docking protocol, a correlation analysis was performed between the computationally derived binding energies (BE) and the experimentally measured IC₅₀ values for the most active compounds (5–8, 18, 19, 22 and 24) and the reference, kojic acid (Figure 4 and Table 2). The resulting correlation is depicted in the scatter plot below.

FIGURE 4.

Correlation between predicted BE and experimental IC₅₀.

The scatter plot illustrates the relationship between the computationally BE (x‐axis) from docking against tyrosinase (PDB: 2Y9X) and the experimentally determined IC₅₀ (y‐axis) for the investigated compounds. The line of best fit is shown in red, and the coefficient of determination (R ²) is displayed. The analysis reveals a strong positive correlation between the predicted binding energies and the observed inhibitory potencies, with a coefficient of determination (R ²) of 0.8754. This value indicates that approximately 87.5% of the variance in the experimental IC₅₀ values can be explained by the predicted binding energy from the docking model. The distinct positive slope of the regression line confirms the expected trend: compounds with more favourable BE consistently exhibit lower IC₅₀ values, signifying higher inhibitory potency. For instance, compound (5), with the most favourable BE of −6.660 kcal/mol, possesses the lowest IC₅₀ of 8.7 µM. Conversely, kojic acid, with the least favourable BE of −4.541 kcal/mol, has one of the highest IC₅₀ values at 18.7 µM. The tight clustering of the data points around the regression line substantiates the reliability of the docking simulations. This strong correlation serves two critical purposes. First, it validates the computational approach, demonstrating that the docking scores are a reliable proxy for biological activity within this chemical series. Second, it reinforces the structural insights derived from the docking poses, confirming that the predicted binding modes and key interactions, particularly the extensive H‐bond networks, are indeed critical drivers of the compounds' inhibitory mechanism against tyrosinase. This robust correlation provides a solid foundation for the structure‐activity relationship discussion and supports the use of this computational model for the future design of even more potent tyrosinase inhibitors based on the studied scaffold.

2.5. Analytical Insights Into Chemical Reactivity Indices of tris‐Schiff Bases as Tyrosinase Inhibitors

Chemical reactivity indices derived from density functional theory (DFT) calculations provide crucial insights into molecular behaviour and biological activity [37]. This analysis examines the reactivity parameters of symmetric bis‐hydrazone tyrosinase inhibitors computed at the CAM‐B3LYP‐D3/MSV level, correlating electronic properties with experimental inhibition (IC₅₀) and docking affinities against PDB:2Y9X. The key global reactivity indices, including the energies of the highest occupied molecular orbital (E_HOMO) and the lowest unoccupied molecular orbital (E_LUMO), the HOMO–LUMO energy gap (E _g), hardness (η), softness (σ), electronegativity (χ) and the electrophilicity index (ω), are presented in Figure 5. The most potent inhibitor, 5 (IC₅₀ = 8.7 µM), possessed the largest energy gap (E _g = 7.258 eV) and consequently the highest chemical hardness (η = 3.629). Conversely, less active compounds, such as the aliphatic derivative 22 (IC₅₀ = 16.2 µM) and the ortho‐chloro derivative 19 (IC₅₀ = 14.2 µM), exhibit the smallest energy gaps (5.564 and 5.978 eV, respectively) and are therefore the softest, most reactive molecules in the series. This establishes a clear positive correlation between chemical stability (high E _g, high η) and inhibitory potency.

FIGURE 5.

Chemical reactivity indices of the synthesised tris‐Schiff bases as tyrosinase inhibitors.

Compound 18, one of the least active inhibitors, has the lowest electrophilicity (ω = 2.259), while the most potent compound 5 has a moderate value (2.471). The compounds with the highest electrophilicity, 19 (3.723) and 22 (3.985), are not the most potent. This indicates that while a degree of electrophilicity is necessary, an excessively high value is not beneficial and may even be detrimental to achieving the optimal interaction profile for potent inhibition.

The HOMO is predominantly localised on the central part of the molecule, encompassing the 2‐fluoro‐[1,1'‐biphenyl] moiety and the adjacent hydrazide linker. This region, rich in electron density, is perfectly positioned to act as a hydrogen bond donor and acceptor, directly corroborating the docking analysis where these groups form critical interactions with the polar/charged residues of the active site. The LUMO, in stark contrast, is almost exclusively located on the two‐terminal para‐nitro benzylidene groups. The strong electron‐withdrawing nature of the nitro groups effectively pulls electron density to the molecular periphery. This distinct separation of electron‐donating and electron‐accepting regions likely facilitates highly specific and directional interactions within the binding pocket, maximising binding affinity. In less active compound 18 (meta‐hydroxy), the HOMO is located on the central core and the hydroxy benzylidene moiety. The LUMO is also distributed across the central region, leading to a less distinct electronic separation compared to compound 5. This less‐defined electronic profile may result in less specific or weaker interactions, consistent with its lower activity. For the nitro isomers, the shift from para (5) to meta (6) and ortho (7) alters the orbital distributions. In compound 7, the steric clash of the ortho‐nitro group likely disrupts the planarity and delocalisation, leading to a less favourable electronic distribution for binding, which is reflected in its higher IC₅₀. For the halogenated compounds 8, 19 and 24, the LUMO is partially localised on the halogen‐substituted ring, but the overall electronic separation is less pronounced than in compound 5. Aliphatic compound 22, lacking an aromatic ring at the terminus, display a LUMO distribution more focused on the central hydrazide backbone. The clear separation of electron‐rich (nucleophilic) and electron‐poor (electrophilic) regions likely facilitates highly specific and directional interactions with the enzyme's active site (Figure 6). This distinct electronic profile was less pronounced in weaker inhibitors.

FIGURE 6.

FMO of the synthesized tris‐Schiff base compounds as tyrosinase inhibitors.

2.6. Analysis of the Correlation Between Molecular Electrostatic Potential (MEP) and IC₅₀

The MEP map of compound 5 provides an ideal template for understanding the requirements for high‐affinity binding (Figure 7). The map is characterised by distinct and intense regions of negative potential (deep red) localised on the oxygen atoms of the two‐terminal para‐nitro groups and, critically, on the carbonyl and ether oxygens of the central scaffold. These red zones represent strong hydrogen bond acceptor sites. Complementarily, regions of positive potential (blue/light blue) are observed around the ─NH protons of the hydrazide linkers, identifying them as potent hydrogen bond donor sites. The strategic positioning of these electron‐rich and electron‐poor zones aligns perfectly with the docking results, which showed these exact moieties forming a dense hydrogen bond network with key residues like Arg164, Glu180 and Asp295. The MEP maps of the meta (6) and ortho nitro (7) isomers highlight the importance of substituent position. While they also display strong negative potentials on the nitro and carbonyl groups, their overall molecular shape and potential distribution are altered. In compound 7, the ortho‐nitro group's proximity to the linker introduces steric strain, twisting the molecule and creating a more convoluted electrostatic surface compared to the well‐defined shape of 5. This suboptimal topography disrupts the ideal electrostatic match with the active site, leading to a weaker binding affinity and higher IC₅₀ value (13.5 µM). The MEP maps for compounds with other electron‐withdrawing or polar groups further refine this understanding.

FIGURE 7.

MEP of the synthesised tris‐Schiff bases as tyrosinase inhibitors.

The hydroxyl‐substituted compound 18 (IC₅₀ = 18.9 µM) shows intense negative (oxygen) and positive (hydrogen) potential at its terminus, but its overall shape and potential distribution lead to the weakest activity among the potent analogues, suggesting a poor geometric and electrostatic fit. The terminal butyl groups (22) are predominantly neutral (green/yellow), lacking the strong negative potential regions seen in the aromatic derivatives. This absence of a key polar interaction site at the molecular termini significantly diminishes its ability to form a comprehensive interaction network, which is reflected in its reduced potency. Generally, we concluded that EWGs (e.g., nitro, chloro, bromo) enhance negative MEP regions, increasing the likelihood of strong interactions with positively charged or H‐bond‐donating residues in the target site. The spatial extension and localisation of negative MEP regions facilitate optimal orientation and binding within the active site, further lowering IC₅₀.

2.7. Analysis of Molecular Dynamics Simulation for Compound 5

To investigate the intrinsic dynamic properties and structural stability of the tyrosinase enzyme upon binding the most potent 5 inhibitor, a normal mode analysis (NMA) was performed. NMA is a computationally efficient method to explore the large‐scale, collective motions of macromolecules that are often functionally relevant. By analysing the deformability, B‐factors, eigenvalues, variance and correlated motions, we can elucidate how the inhibitor modulates the enzyme's dynamics to achieve its potent inhibitory effect (Figure 8). These findings bridge the gap between static docking predictions and biologically relevant protein–ligand interactions, offering a mechanistic framework for rational drug design. Figure 8 dissects the critical insights derived from the simulation data and correlates them with experimental potency. Deformability profiles displaying the low atomic fluctuations (<0.2 Å) in the central domain (residues 600–1200) contrast sharply with the inherent flexibility of the unbound enzyme. This stabilisation likely restricts the conformational sampling required for substrate turnover, effectively “locking” the enzyme in an inactive state. The reduction in B‐factors (NMA vs. PDB: 2Y9X) comparison underscores compound 5's ability to suppress thermal motions, enhancing structural rigidity. This aligns with its experimental potency, as rigid binding geometries often correlate with prolonged residence times and stronger inhibition. The active site rigidity imposed by compound 5 mirrors the effects of covalent inhibitors, albeit through non‐covalent interactions. This dynamic stabilisation likely disrupts the enzyme's catalytic cycle, preventing the oxidation of tyrosine to dopaquinone, a critical step in melanogenesis.

FIGURE 8.

The MD for compound 5 at 100 ns, including deformability profiles, B‐factor comparisons, eigenvalue distributions, 3D structural flexibility visualisation, residue interaction maps and cross‐correlation matrices.

The residue interaction map highlights dense red clusters, which represent strong inter‐residue couplings (e.g., Glu180‐Arg164, Thr306‐Asp295) within the active site, indicative of H‐bond networks and salt bridges. The interactions at the active site (residues 200–800) and distant regions (1200–1600) suggest that the binding of compound 5 influences the dynamics of distant regions, indicating long‐range stabilisation, propagating stability across the protein. The tight packing of residues around compound 5 likely disrupts the mobility of Cu²⁺ coordinating histidines (His61, His85, His94), which are essential for tyrosinase activity. This long‐range stabilization combined with direct active site binding, likely contributes to the potent inhibition of tyrosinase activity. The eigenvalue analysis of collective motions reveals low‐frequency dominance with ∼90% of variance captured by the first 20 modes, dominated by rigid‐body‐like movements (e.g., domain rotations). While the suppressed flexibility for compound 5 dampens large‐scale motions (eigenvalues < 0.1), it reduces the entropy penalty associated with binding. The suppression of low‐frequency motions suggests that compound 5 optimises the enthalpy–entropy trade‐off during binding. By minimising conformational entropy loss, it achieves tighter binding compared to less rigid inhibitors like kojic acid. The inset MD data for kojic acid contrasts with compound 5. Higher B‐Factors (Figure 8), greater atomic mobility in the active site (B‐factors > 0.5) indicate weaker binding and faster dissociation. While loose residue packing, displaying sparse interaction clusters, suggests limited cooperation between active site residues, it reduces inhibitory efficacy. Kojic acid's instability underscores the importance of dynamic complementarity, wherein inhibitors must adapt to and stabilise the enzyme's native dynamics. Compound 5 exemplifies this principle, whereas kojic acid's planar structure fails to engage the protein's flexible regions effectively. The cross‐correlation matrix reveals localised flexibility, represents strong positive correlations within structural domains (red diagonal) versus weak inter‐domain couplings (blue off‐diagonal), suggesting compartmentalised dynamics. The residues 600–1200 exhibit synchronised motions, reinforcing the hypothesis that compound 5 stabilises a functionally inactive conformation.

The 3D flexibility map correlates spatially with activity; the active site's rigidity (blue‐rigid regions) ensures precise positioning of compound 5’s nitro benzylidene and hydrazide moieties, enabling optimal hydrogen bonding and π–π stacking. The peripheral flexibility (yellow/orange, flexible loops) minimises steric clashes, allowing the inhibitor to adapt to minor conformational shifts without losing affinity. The para‐nitro substitution on the benzylidene ring likely enhances this dynamic fit by extending conjugation and strengthening electrostatic interactions with Arg164, a critical residue for substrate recognition.

3. Experimental Section

3.1. General

In this research work, all the chemicals, solvents and reagents were of analytical grade (99% pure) and procured from Sigma Aldrich and TCI. Using silica gel 60F‐254 from Merck, Darmstadt, Hesse, Germany, thin‐layer chromatography (TLC) was carried out on pre‐coated aluminium sheets. The results were visualised under UV light at a wavelength of 254 nm. The Stuart SMP10 device was used to ascertain the melting points of the synthetic equivalents. The nuclear magnetic resonance (NMR, Zürich, Switzerland's Bruker) spectroscopy was recorded for ¹H at 600 MHz and for ¹³C at 150 MHz, while the chemical shift (δ) was reported in parts per million (ppm), and the coupling constants (J) in Hertz (Hz).

3.2. Procedure for the Synthesis of Compounds (2–4)

The commercially available flurbiprofen was first coupled with hydrazine hydrate in THF solvent containing 1,1‐carbonyldiimidazole (CDI), and the reaction was refluxed for 3–4 h while it was being constantly stirred to get the hydrazide in better yield. Subsequently, the obtained hydrazide of flurbiprofen was further refluxed with diethyl 2,2'‐((4‐formyl‐1,3‐phenylene) bis(oxy)) diacetate in absolute ethanol having a minute amount of acetic acid for 4‐5 h to get the esterified Schiff base compound 3 in better yield. Finally, the desired compound 3 was treated with hydrazine hydrate in ethanol at reflux for 4–5 h to get the bis‐Schiff base hydrazide (4) in good yield. The thin‐layer chromatographic technique was utilised to determine the formation of the product compounds. After the completion, reaction mixtures were transferred into a beaker containing ice‐cold distilled water; the precipitates that appeared were filtered, washed with distilled water and dried overnight. These compounds were recrystallised through pure ethanol to get them in pure form. Structures of these derivatives were thoroughly confirmed by means of HR‐ESI‐MS, ¹H‐ and ¹³C‐NMR spectroscopy.

3.3. General Procedure for the Synthesis of tris‐Schiff Base Derivatives (5–25)

The synthesised bis‐hydrazide Schiff base (4) of flurbiprofen was refluxed for 3–4 h with aliphatic and different substituted aromatic aldehydes in the presence of a catalytic quantity of acetic acid in absolute ethanol to get the tris‐Schiff bases (5–24) in significant yields. The TLC technique was employed to determine the formation of product compounds. After the reactions were completed, these were transferred to a beaker containing cold distilled water; the coloured precipitates formed were filtered, washed with hot water, dried overnight and collected. The obtained tris‐Schiff base compounds were recrystallised through modern spectroscopic techniques like HR‐ESI‐MS, ¹³C‐ and ¹H‐NMR (Supporting Information).

3.4. Spectral Interpretation of the Compounds (4–24)

3.4.1. 2,2'‐((4‐((2‐(2‐(2‐Fluoro‐[1,1'‐biphenyl]‐4‐yl)propanoyl)hydrazineylidene)methyl)‐1,3‐phenylene)bis(oxy))di(aceto hydrazide) (4)

Yield: 72%; ¹H‐NMR (600 MHz, MeOD): δ 9.48 (br.s, 1H, ─NH), 8.11 (s, 1H, H‐16), 7.53–7.50 (m, 5H, Ar‐H, H‐8‐12), 7.38–7.35 (m, 2H, Ar‐H, H‐5, H‐6), 7.25–7.20 (m, 2H, Ar‐H, H‐2, H‐6'), 6.66 (d, J = 8.4 Hz, 1H, Ar‐H, H‐5'), 6.62 (br.s, 1H, Ar‐H, H‐5'), 4.90 (br.s, 4H, 2‐NH₂), 4.59 (s, 4H, H‐17, H‐18), 3.52–3.48 (m, 1H, H‐13), 1.50 (d, J = 7.2 Hz, 3H, H‐15). ¹³C‐NMR (150 MHz, MeOD): δ 175.7 (C‐14), 161.7 (2C═O), 160.8 (C‐4'), 160.1 (C‐2'), 157.7 (C‐3), 144.4 (C‐16), 136.9 (C‐1, C‐7), 131.8 (C‐6'), 129.9 (C‐5, C‐9, C‐11), 129.5 (C‐10), 128.9 (C‐4), 128.8 (C‐8, C‐12), 124.8 (C‐6), 116.1 (C‐2), 115.9 (C‐5'), 108.7 (C‐1'), 101.8 (C‐3'), 68.3 (C‐17), 67.7 (C‐18), 45.1 (C‐13), 18.6 (C‐15). HRMS (ESI⁺): [M + H]+ calcd for C₂₆H₂₇FN₆O₅: 522.2027; found 522.2937.

3.4.2. 2,2'‐((4‐(2‐(2‐(2‐Fluoro‐[1,1'‐biphenyl]‐4‐yl)propanoyl)hydrazono)methyl)‐1,3‐phenylene)bis(oxy))bis(N'‐(4‐nitro benzylidene)acetohydrazide) (5)

Yield: 80%; ¹H‐NMR (600 MHz, MeOD): δ 8.78 (s, 1H, H‐16), 8.54 (s, 2H, H‐17, H‐18), 8.37–8.24 (m, 4H, Ar‐H, H‐3″, H‐5″, H‐3″′, H‐5″′), 8.16–8.14 (m, 1H, Ar‐H, H‐5), 8.02 (d, J = 6.6 Hz, 2H, Ar‐H, H‐2″, H‐6″), 7.92 (d, J = 8.4 Hz, 1H, Ar‐H, H‐6), 7.52 (d, J = 7.8 Hz, 2H, Ar‐H, H‐2″′, H‐6″′), 7.49–7.41 (m, 5H, Ar‐H, H‐8‐10), 7.40–7.25 (m, 4H, Ar‐H, H‐2, H‐3', H‐5', H‐6'), 4.64 (s, 4H, H‐19, H‐20), 3.85–3.81 (m, 1H, H‐13), 1.59 (d, J = 7.2 Hz, 3H, H‐15). ¹³C‐NMR (150 MHz, MeOD): δ 173.5 (C‐14), 161.8 (2C═O), 160.2 (C‐3), 150.0 (C‐4″, C‐4″′), 149.8 (C‐4'), 147.1 (C‐17, C‐18), 144.0 (C‐2'), 143.1 (C‐16), 141.6 (C‐1″, C‐1″′), 136.8 (C‐1, C‐7), 132.1 (C‐5), 131.9 (C‐6'), 129.9 (C‐9, C‐11), 129.1 (C‐4), 128.9 (C‐3″, C‐5″, C‐3″′, C‐5″′), 128.8 (C‐8, C‐12), 128.7 (C‐10), 125.1 (C‐2″, C‐6″, C‐2″′, C‐6″′), 124.9 (C‐5′), 124.8 (C‐6), 116.4 (C‐1′), 116.2 (C‐3′), 116.0 (C‐2), 69.0 (C‐19, C‐20), 45.7 (C‐13), 19.1 (C‐15). HRMS (ESI⁺): [M + H]⁺ calcd for C₄₀H₃₃FN₈O₉: 788.2355; found 788.0410.

3.4.3. 2,2′‐((4‐(2‐(2‐(2‐Fluoro‐[1,1′‐biphenyl]‐4‐yl)propanoyl)hydrazineylidene)methyl)‐1,3‐phenylene)bis(oxy))bis(N'‐(3‐nitrobenzylidene)acetohydrazide) (6)

Yield: 78%; ¹H‐NMR (600 MHz, MeOD): δ 8.82 (s, 2H, H‐17, H‐18), 8.79 (s, 1H, H‐16), 8.52 (s, 2H, Ar‐H, H‐2″, H‐2″′), 8.39 (d, J = 8.4 Hz, 1H, Ar‐H, H‐5), 8.30–8.25 (m, 3H, Ar‐H, H‐9, H‐10, H‐11), 8.19 (d, J = 7.8 Hz, 1H, Ar‐H, H‐6), 8.00 (s, 1H, Ar‐H, H‐2), 7.77 (t, J = 7.8 Hz, 2H, Ar‐H, H‐5″, H‐5'″), 7.70–7.66 (m, 1H, Ar‐H, H‐6'), 7.53 (d, J = 7.8 Hz, 1H, Ar‐H, H‐5'), 7.50–7.40 (m, 4H, Ar‐H, H‐4″, H‐6″, H‐4″′, H‐6″′), 7.38–7.30 (m, 2H, Ar‐H, H‐8, H‐12), 7.29–7.22 (m, 1H, Ar‐H, H‐3'), 4.64 (s, 4H, H‐19, H‐20), 3.84–3.80 (m, 1H, H‐13), 1.59 (d, J = 6.6 Hz, 3H, H‐15). ¹³C‐NMR (150 MHz, MeOD): δ 173.5 (C‐14), 161.9 (2C═O), 160.1 (C‐3), 150.1 (C‐4'), 147.2 (C‐3″, C‐3″′), 143.0 (C‐17, C‐18), 144.0 (C‐2'), 137.7 (C‐1), 136.8 (C‐7), 135.5 (C‐16), 133.8 (C‐1″, C‐1″′), 132.0 (C‐6″, C‐6″′), 131.9 (C‐6'), 131.4 (C‐5), 130.0 (C‐9, C‐11), 129.9 (C‐5″, C‐5″′), 129.5 (C‐8, C‐12), 128.8 (C‐10), 128.7 (C‐4), 126.8 (C‐4″, C‐4″′), 124.8 (C‐6), 123.8 (C‐2″, C‐2″′), 123.2 (C‐5'), 122.3 (C‐1'), 116.2 (C‐3'), 116.0 (C‐2), 69.0 (C‐19, C‐20), 45.7 (C‐13), 19.1 (C‐15). HRMS (ESI⁺): [M + H]⁺ calcd for C₄₀H₃₅FN₈O₉: 790.2355; found 790.1311.

3.4.4. 2,2'‐((4‐(2‐(2‐(2‐Fluoro‐[1,1'‐biphenyl]‐4‐yl)propanoyl)hydrazono)methyl)‐1,3‐phenylene)bis(oxy))bis(N'‐(2‐nitro benzylidene)acetohydrazide) (7)

Yield: 76%; ¹H‐NMR (600 MHz, DMSO‐d₆ ): δ 9.58 (br.s, 1H, ─NH), 9.39 (br.s, 2H, 2‐NH), 8.94 (s, 2H, H‐17, H‐18), 8.59 (s, 1H, H‐16), 8.31 (s, 1H, Ar‐H, H‐2), 8.14 (d, J = 7.8 Hz, 3H, Ar‐H, H‐5, H‐6″, H‐6″′), 8.05–7.93 (m, 4H, Ar‐H, H‐4″, H‐4″′, H‐8, H‐12), 7.86 (t, J = 7.2 Hz, 2H, Ar‐H, H‐5″, H‐5'″), 7.79–7.77 (m, 4H, Ar‐H, H‐6, H‐9‐11), 7.65–7.60 (m, 2H, Ar‐H, H‐3″, H‐3'″), 7.40–7.34 (m, 1H, Ar‐H, H‐6'), 6.68–6.59 (m, 2H, Ar‐H, H‐3', H‐5'), 4.71–4.45 (m, 4H, H‐19, H‐20), 3.78‐3.76 (m, 1H, H‐13), 1.44–1.41 (m, 3H, H‐15). ¹³C‐NMR (150 MHz, DMSO‐d₆ ): δ 174.8 (C‐14), 169.8 (2C═O), 166.3 (C‐4'), 159.7 (C‐3), 158.7 (C‐17, C‐18), 158.1 (C‐2'), 148.1 (C‐2″, C‐2″′), 142.4 (C‐16), 138.6 (C‐1, C‐7), 134.0 (C‐5″, C‐5″′), 132.9 (C‐6'), 132.2 (C‐4″, C‐4″′), 130.7 (C‐5), 129.5 (C‐6″, C‐6″′), 128.7 (C‐9, C‐11), 128.6 (C‐8, C‐12), 127.8 (C‐10), 127.5 (C‐4), 126.8 (C‐1″, C‐1″′), 124.8 (C‐3″, C‐3″′), 124.7 (C‐6), 124.1 (C‐5'), 123.9 (C‐3'), 114.9 (C‐2), 109.2 (C‐1'), 66.4 (C‐19, C‐20), 43.6 (C‐13), 18.4 (C‐15). HRMS (ESI⁺): [M + 2H]⁺ calcd for C₄₀H₃₅FN₈O₉: 790.2461; found 791.3919.

3.4.5. 2,2'‐((4‐(2‐(2‐(2‐Fluoro‐[1,1'‐biphenyl]‐4‐yl)propanoyl)hydrazono)methyl)‐1,3‐phenylene)bis(oxy))bis(N'‐(4‐chloro benzylidene)acetohydrazide) (8)

Yield: 75%; ¹H‐NMR (600 MHz, DMSO): δ 11.38 (s, 2H, 2‐NH), 11.16 (s, 1H, ─NH), 8.02 (s, 1H, H‐16), 7.79 (s, 2H, H‐19, H‐20), 7.49 (t, J = 8.0 Hz, 4H, Ar‐H, H‐5, H‐9, H‐10, H‐11), 7.45–7.41 (m, 4H, Ar‐H, H‐2″, H‐6″, H‐2″′, H‐6″′), 7.37–7.33 (m, 2H, Ar‐H, H‐8, H‐12), 7.26 (d, J = 8.4 Hz, 4H, Ar‐H, H‐3″, H‐5″, H‐3″′, H‐5″′), 7.19–7.16 (m, 2H, Ar‐H, H‐2, H‐6'), 6.99–6.96 (m, 2H, Ar‐H, H‐3', H‐6), 6.93 (d, J = 7.8 Hz, 1H, Ar‐H, H‐5'), 4.71–4.54 (m, 4H, H‐17, H‐18), 3.76–3.72 (m, 1H, H‐13), 1.41 (d, J = 6.2 Hz, 3H, H‐15). ¹³C‐NMR (150 MHz, DMSO) δ 174.0 (C‐14), 168.9 (2C═O), 159.6 (C‐4'), 158.0 (C‐3), 149.5 (C‐2'), 146.8 (C‐19, C‐20), 143.8 (C‐1), 143.3 (C‐16), 134.9 (C‐4″, C‐4″′), 130.6 (C‐5), 130.6 (C‐7), 128.7 (C‐3″, C‐5″, C‐3″′, C‐5″′), 128.6 (C‐9, C‐11), 128.5 (C‐2″, C‐6″, C‐2″′, C‐6″′), 127.8 (C‐4), 127.0 (C‐8, C‐12), 126.9 (C‐10), 126.6 (C‐6), 124.1 (C‐1″, C‐1″′), 120.2 (C‐6'), 119.8 (C‐2), 115.2 (C‐5'), 114.8 (C‐3'), 111.8 (C‐1'), 66.4 (C‐17, C‐18), 43.4 (C‐13), 18.2 (C‐15). HRMS (ESI⁺): [M + H]⁺ calcd for C₄₀H₃₃Cl₂FN₆O₅: 767.6394; found 767.4895.

3.4.6. 2,2'‐((4‐(2‐(2‐(2‐Fluoro‐[1,1'‐biphenyl]‐4‐yl)propanoyl)hydrazono)methyl)‐1,3‐phenylene)bis(oxy))bis(N'‐(2‐methoxy benzylidene)acetohydrazide) (9)

Yield: 70%; ¹H‐NMR (600 MHz, DMSO‐d₆ ): δ 8.26 (s, 1H, H‐16), 8.54 (s, 2H, H‐17, H‐18), 8.37–8.24 (m, 4H, Ar‐H, H‐3″, H‐5″, H‐3″′, H‐5″′), 8.16–8.14 (m, 1H, Ar‐H, H‐5), 8.02 (d, J = 6.6 Hz, 2H, Ar‐H, H‐6″, H‐6″′), 7.92 (d, J = 8.4 Hz, 1H, Ar‐H, H‐6), 7.52–7.50 (m, 5H, Ar‐H, H‐2, H‐9, H‐11, H‐3', H‐6'), 7.49 (d, J = 7.2 Hz, 2H, Ar‐H, H‐8, H‐12), 7.22 (t, J = 8.4 Hz, 1H, Ar‐H, H‐10), 6.93 (d, J = 7.8 Hz, 1H, Ar‐H, H‐5'), 6.83 (t, J = 7.2 Hz, 2H, Ar‐H, H‐4″, H‐4″′), 4.64–4.53 (m, 4H, H‐19, H‐20), 3.78–3.76 (m, 1H, H‐13), 3.71 (s, 6H, 2‐OCH₃), 1.59 (d, J = 7.2 Hz, 3H, H‐15). ¹³C‐NMR (150 MHz, DMSO‐d₆ ): δ 174.4 (C‐14), 169.2 (2C═O), 163.8 (C‐4'), 159.7 (C‐3), 158.1 (C‐2'), 157.6 (C‐2″, C‐2″′), 145.9 (C‐16), 142.9 (C‐17, C‐18), 142.0 (C‐1'), 136.9 (C‐7), 134.9 (C‐1), 131.7 (C‐6″, C‐6″′), 129.9 (C‐5), 129.4 (C‐6'), 129.2 (C‐9, C‐11), 128.6 (C‐8, C‐12), 127.8 (C‐4, C‐10), 124.1 (C‐6), 123.8 (C‐5'), 121.1 (C‐5″, C‐5″′), 119.6 (C‐3″, C‐3″′), 116.9 (C‐1″′, C‐1″′), 115.7 (C‐3'), 114.9 (C‐2), 112.0 (C‐4″, C‐4″′), 66.4 (C‐19, C‐20), 55.6 (2‐OCH₃), 43.5 (C‐13), 18.3 (C‐15). HRMS (ESI⁺): [M + H]⁺ calcd for C₄₂H₃₉FN₆O₇: 758.2864; found 758.1956.

3.4.7. 2,2'‐((4‐(2‐(2‐(2‐Fluoro‐[1,1'‐biphenyl]‐4‐yl)propanoyl)hydrazono)methyl)‐1,3‐phenylene)bis(oxy))bis(N'‐(3‐methoxy benzylidene)acetohydrazide) (10)

Yield: 67%; ¹H‐NMR (600 MHz, DMSO‐d₆ ): δ 11.59 (s, 2H, 2‐NH), 11.36 (s, 1H, ─NH), 8.66 (s, 2H, H‐17, H‐18), 8.16 (s, 1H, H‐16), 7.92–7.91 (m, 1H, Ar‐H, H‐5), 7.88 (s, 1H, Ar‐H, H‐2), 7.45–7.38 (m, 6H, Ar‐H, H‐4″, H‐5″, H‐6″, H‐4″′, H‐5″′, H‐6″′), 7.37–7.31 (m, 2H, Ar‐H, H‐8, H‐12), 7.30–7.27 (m, 2H, Ar‐H, H‐2″, H‐2'″), 7.25–7.18 (m, 3H, Ar‐H, H‐9‐11), 7.08–7.04 (m, 1H, Ar‐H, H‐6), 6.98–6.93 (m, 2H, Ar‐H, H‐5', H‐6'), 6.64–6.63 (m, 1H, Ar‐H, H‐3'), 4.81‐4.54 (m, 4H, H‐19, H‐20), 3.79 (s, 3H, ─OCH₃), 3.75 (s, 3H, ─OCH₃), 3.74–3.71 (m, 1H, H‐13), 1.40 (d, J = 7.2 Hz, 3H, H‐15). ¹³C‐NMR (150 MHz, DMSO‐d₆ ): δ 174.4 (C‐14), 169.2 (2C═O), 161.5 (C‐3″, C‐3″′), 159.5 (C‐3), 158.0 (C‐4'), 146.9 (C‐17, C‐18), 144.0 (C‐2'), 143.9 (C‐16), 135.6 (C‐1″, C‐1″′), 135.3 (C‐1), 134.9 (C‐7), 130.7 (C‐6'), 130.0 (C‐5), 129.9 (C‐9, C‐11), 128.7 (C‐8, C‐12), 128.6 (C‐5″, C‐5″′), 127.7 (C‐10), 126.7 (C‐4), 126.4 (C‐1'), 123.9 (C‐6), 123.8 (C‐5'), 121.3 (C‐6″), 121.2 (C‐6″′), 116.2 (C‐4″), 116.1 (C‐4″′), 115.2 (C‐3'), 114.9 (C‐2), 111.3 (C‐2″, C‐2″′), 66.4 (C‐19, C‐20), 55.2 (2‐OCH₃), 43.5 (C‐13), 18.4 (C‐15). HRMS (ESI⁺): [M + H]⁺ calcd for C₄₂H₃₉FN₆O₇: 758.2864; found 758.1956.

3.4.8. 2,2'‐((4‐(2‐(2‐(2‐Fluoro‐[1,1'‐biphenyl]‐4‐yl)propanoyl)hydrazono)methyl)‐1,3‐phenylene) bis(oxy))bis(N'‐(2,4‐dimethoxy benzylidene)acetohydrazide) (11)

Yield: 66%; ¹H‐NMR (600 MHz, DMSO‐d₆ ): δ 11.42 (s, 1H, ─NH), 9.55 (s, 2H, 2‐NH), 8.92 (s, 2H, H‐17, H‐18), 8.42 (s, 1H, H‐16), 8.17‐8.50 (m, 3H, Ar‐H, H‐3″, H‐8, H‐12), 8.49‐8.33 (m, 6H, Ar‐H, H‐5″, H‐6″, H‐5″′, H‐6″′, H‐9, H‐11), 7.26–7.21 (m, 2H, Ar‐H, H‐3″′, H‐10), 6.67–6.63 (m, 6H, Ar‐H, H‐2, H‐5, H‐6, H‐3', H‐5', H‐6'), 4.59 (s, 2H, H‐19), 4.54 (s, 2H, H‐20), 3.81 (s, 6H, 2‐OCH₃), 3.78 (s, 6H, 2‐OCH₃), 3.76‐3.74 (m, 1H, H‐13), 1.41‐1.38 (m, 3H, H‐15). ¹³C‐NMR (150 MHz, DMSO‐d₆ ): δ 174.2 (C‐14), 168.7 (2C═O), 162.4 (C‐2″, C‐2″′), 162.2 (C‐4'), 159.1 (C‐3), 158.9 (C‐4″, C‐4″′), 158.2 (C‐2'), 142.4 (C‐17, C‐18), 138.8 (C‐16), 134.9 (C‐1, C‐7), 129.5 (C‐5), 128.7 (C‐6'), 128.6 (C‐9, C‐11, C‐6″, C‐6″′), 127.8 (C‐8, C‐12), 127.7 (C‐10), 126.5 (C‐4), 126.6 (C‐5'), 123.8 (C‐6), 115.1 (C‐3'), 114.9 (C‐2), 107.8 (C‐1″, C‐1″′), 106.5 (C‐1'), 106.4 (C‐5″, C‐5″′), 98.3 (C‐3″′), 98.2 (C‐3″), 67.0 (C‐19, C‐20), 55.8 (2‐OCH₃), 55.4 (2‐OCH₃), 43.5 (C‐13), 18.3 (C‐15). HRMS (ESI⁺): [M + H]⁺ calcd for C₄₄H₄₃FN₆O₉: 819.3076; found 819.8627.

3.4.9. 2,2'‐((4‐(2‐(2‐(2‐Fluoro‐[1,1'‐biphenyl]‐4‐yl)propanoyl)hydrazono)methyl)‐1,3‐phenylene)bis(oxy))bis(N'‐(3‐hydroxy‐4‐methoxybenzylidene)acetohydrazide) (12)

Yield: 64%; ¹H‐NMR (600 MHz, DMSO‐d₆ ): δ 11.38 (s, 2H, 2‐NH), 9.25 (s, 2H, 2‐OH), 8.07 (s, 1H, H‐16), 8.02 (s, 2H, H‐17, H‐18), 7.50–7.40 (m, 7H, Ar‐H, H‐2″, H‐5″, H‐6″, H‐2″′, H‐5″′, H‐6″′, H‐5), 7.37–7.33 (m, 2H, Ar‐H, H‐2, H‐6), 7.26–7.15 (m, 5H, Ar‐H, H‐8‐12), 6.98 (br.s, 1H, Ar‐H, H‐3'), 6.91–6.90 (m, 1H, Ar‐H, H‐6'), 6.66 (br.s, 1H, Ar‐H, H‐5'), 4.71‐4.44 (m, 4H, H‐19, H‐20), 3.79 (s, 6H, 2‐OCH₃), 3.72‐3.70 (m, 1H, H‐13), 1.41‐1.36 (m, 3H, H‐15). ¹³C‐NMR (150 MHz, DMSO‐d₆ ): δ 174.1 (C‐14), 168.9 (2C═O), 159.7 (C‐3), 158.0 (C‐4″, C‐4″′), 149.8 (C‐4'), 149.5 (C‐2'), 147.1 (C‐17, C‐18), 146.8 (C‐3″, C‐3″′), 143.3 (C‐16), 134.9 (C‐1, C‐1″, C‐1″′, C‐7), 130.7 (C‐5), 130.6 (C‐9, C‐11), 128.7 (C‐6'), 128.6 (C‐8, C‐12), 127.7 (C‐10), 126.6 (C‐4), 126.4 (C‐1'), 124.2 (C‐6), 123.8 (C‐5'), 120.2 (C‐6″, C‐6″′), 115.2 (C‐2″, C‐2″′), 115.1 (C‐3'), 114.8 (C‐2), 111.9 (C‐5″, C‐5″′), 67.0 (C‐19, C‐20), 55.6 (2‐OCH₃), 43.5 (C‐13), 18.2 (C‐15). HRMS (ESI⁺): [M +H]⁺ calcd for C₄₂H₃₉FN₆O₉: 790.2763; found 790.0025.

3.4.10. 2,2'‐((4‐(2‐(2‐(2‐Fluoro‐[1,1'‐biphenyl]‐4‐yl)propanoyl)hydrazono)methyl)‐1,3‐phenylene)bis(oxy))bis(N'‐(2‐hydroxy‐3‐methoxybenzylidene)acetohydrazide) (13)

Yield: 64%; ¹H‐NMR (600 MHz, DMSO‐d₆ ): δ 11.75 (s, 2H, 2‐NH), 10.68 (s, 1H, ─NH), 9.32 (br.s, 2H, 2‐OH), 8.95 (s, 1H, H‐16), 8.38 (s, 2H, H‐17, H‐18), 7.50–7.34 (m, 6H, Ar‐H, H‐4″, H‐5″, H‐6″, H‐4″′, H‐5″′, H‐6″′), 7.28–7.25 (m, 3H, Ar‐H, H‐9‐11), 7.25–7.06 (m, 4H, Ar‐H, H‐5, H‐6, H‐8, H‐12), 6.98–6.78 (m, 4H, Ar‐H, 2H, H‐3', H‐5', H‐6'), 4.65‐4.54 (m, 4H, H‐19, H‐20), 3.76 (s, 6H, 2‐OCH₃), 3.73‐3.71 (m, 1H, H‐13), 1.43‐1.33 (m, 3H, H‐15). ¹³C‐NMR (150 MHz, DMSO‐d₆ ): δ 174.1 (C‐14), 169.0 (2C═O), 159.7 (C‐3), 158.1 (C‐2″, C‐2″′), 148.5 (C‐3″, C‐3″′), 148.0 (C‐4'), 147.2 (C‐17, C‐18), 145.9 (C‐2'), 140.6 (C‐16), 134.9 (C‐1, C‐7), 130.8 (C‐5), 130.7 (C‐9, C‐11), 128.7 (C‐6'), 128.6 (C‐8, C‐12), 127.7 (C‐10), 126.8 (C‐4), 126.3 (C‐1'), 124.0 (C‐6), 123.9 (C‐6″, C‐6″′), 122.0 (C‐5'), 119.3 (C‐5″, C‐5″′), 118.9 (C‐1″, C‐1″′), 115.3 (C‐3'), 115.1 (C‐4″, C‐4″′), 114.9 (C‐2), 66.4 (C‐19, C‐20), 55.9 (2‐OCH₃), 43.4 (C‐13), 18.2 (C‐15). HRMS (ESI⁺): [M + H]⁺ calcd for C₄₂H₃₉FN₆O₉: 790.2763; found 791.3004.

3.4.11. 2,2'‐((4‐(2‐(2‐(2‐fluoro‐[1,1'‐biphenyl]‐4‐yl)propanoyl)hydrazono)methyl)‐1,3‐phenylene)bis(oxy))bis(N'‐(3,4‐dimethoxy benzylidene)acetohydrazide) (14)

Yield: 62%; ¹H‐NMR (600 MHz, DMSO‐d₆ ): δ 11.44 (br.s, 2H, 2‐NH), 9.55 (s, 1H, ─NH), 8.932 (s, 1H, H‐16), 8.10 (s, 2H, H‐17, H‐18), 7.50–7.40 (m, 8H, Ar‐H, H‐2″, H‐5″, H‐6″, H‐2″′, H‐5″′, H‐6″′, H‐8, H‐12), 7.38–7.33 (m, 3H, Ar‐H, H‐9‐11), 7.28–7.23 (m, 2H, Ar‐H, H‐5, H‐6), 7.14–7.08 (m, 2H, Ar‐H, H‐2, H‐5'), 6.98–6.66 (m, 2H, Ar‐H, H‐2', H‐4'), 4.69‐4.54 (m, 4H, H‐19, H‐20), 3.82 (s, 6H, 2‐OCH₃), 3.75 (s, 6H, 2‐OCH₃), 3.73‐3.71 (m, 1H, H‐13), 1.39 (d, J = 6.6 Hz, 3H, H‐15). ¹³C‐NMR (150 MHz, DMSO‐d₆ ): δ 174.1 (C‐14), 169.0 (2C═O), 166.4 (C‐4'), 159.2 (C‐3), 158.0 (C‐2'), 150.4 (C‐4″, C‐4″′), 149.1 (C‐3″, C‐3″′), 147.2 (C‐17, C‐18), 142.9 (C‐16), 136.9 (C‐7), 134.9 (C‐1, C‐1″, C‐1″′), 130.7 (C‐5), 128.7 (C‐9, C‐11), 128.6 (C‐8, C‐12), 127.8 (C‐10), 127.7 (C‐6'), 127.0 (C‐4), 126.7 (C‐1'), 123.9 (C‐6), 121.8 (C‐6″′), 121.3 (C‐6″), 115.3 (C‐3'), 114.8 (C‐2), 111.5 (C‐2″, C‐2″′), 108.2 (C‐5'), 107.9 (C‐5″), 107.8 (C‐5″′), 66.4 (C‐19, C‐20), 55.6 (2‐OCH₃), 55.4 (2‐OCH₃), 43.5 (C‐13), 18.5 (C‐15). HRMS (ESI⁺): [M + H]⁺ calcd for C₄₄H₄₃FN₆O₉: 818.3076; found 819.8627.

3.4.12. 2,2'‐((4‐(2‐(2‐(2‐fluoro‐[1,1'‐biphenyl]‐4‐yl)propanoyl)hydrazono)methyl)‐1,3‐phenylene)bis(oxy))bis(N'‐(2‐hydroxy benzylidene)acetohydrazide) (15)

Yield: 68%; ¹H‐NMR (600 MHz, DMSO‐d₆ ): δ 9.57 (br.s, 1H, ‐OH), 9.36 (br.s, 1H, ‐OH), 8.97 (s, 2H, H‐17, H‐18), 8.38 (s, 1H, H‐16), 7.87–7.83 (m, 1H, Ar‐H, H‐5), 7.67–7.50 (m, 2H, Ar‐H, H‐8, H‐12), 7.49–7.40 (m, 6H, Ar‐H, H‐3″, H‐4″, H‐5″, H‐3″′, H‐4″′, H‐5″′), 7.38–7.34 (m, 2H, Ar‐H, H‐6″, H‐6″′), 7.28–7.19 (m, 4H, Ar‐H, H‐6, H‐9‐11), 6.96–6.92 (m, 3H, Ar‐H, H‐2, H‐5', H‐6'), 6.66 (br.s, 1H, Ar‐H, H‐3'), 4.66‐4.55 (m, 4H, H‐19, H‐20), 3.76‐3.72 (m, 1H, H‐13), 1.44 (d, J = 6.6 Hz, 3H, H‐15). ¹³C‐NMR (150 MHz, DMSO‐d₆ ): δ 174.4 (C‐14), 169.2 (2 C═O), 163.8 (C‐4'), 159.7 (C‐3), 158.7 (C‐2″, C‐2″′), 158.1 (C‐2'), 146.0 (C‐17, C‐18), 145.9 (C‐16), 142.0 (C‐1'), 136.9 (C‐7), 134.9 (C‐1), 132.4 (C‐4″, C‐4″′), 129.4 (C‐6'), 129.2 (C‐9, C‐11), 128.9 (C‐5), 128.7 (C‐6″, C‐6″′), 128.6 (C‐8, C‐12), 127.8 (C‐4, C‐10), 124.1 (C‐6), 123.8 (C‐5'), 121.4 (C‐5″, C‐5″′), 118.5 (C‐1″, C‐1″′), 117.8 (C‐3″, C‐3″′), 115.7 (C‐3'), 114.9 (C‐2), 66.4 (C‐19, C‐20), 43.5 (C‐13), 18.3 (C‐15). HRMS (ESI⁺): [M + NH₄]⁺ calcd for C₄₀H₃₉FN₇O₇: 748.2890; found 748.2648.

3.4.13. 2‐(2‐fluoro‐[1,1'‐biphenyl]‐4‐yl)‐N'‐(4‐(2‐(2‐(5‐methylfuran‐2‐yl)methylene)hydrazinyl)‐2‐oxoethyl)‐2‐((2‐(2‐(5‐methylfuran‐2‐yl)methylene)hydrazinyl)‐2‐oxoethyl)peroxy)benzylidene)propanehydrazide) (16)

Yield: 70%; ¹H‐NMR (600 MHz, DMSO‐d₆ ): δ 11.43 (br.s, 2H, 2‐NH), 9.36 (s, 1H, ─NH), 8.93 (s, 1H, H‐16), 8.08 (s, 2H, H‐17, H‐18), 7.96–7.74 (m, 4H, Ar‐H, H‐2″, H‐3″, H‐2″′, H‐3″′), 7.50–7.41 (m, 3H, Ar‐H, H‐9‐11), 7.37–7.34 (m, 1H, Ar‐H, H‐5), 7.26–7.22 (m, 1H, Ar‐H, H‐6), 6.94–6.64 (m, 4H, Ar‐H, H‐2, H‐8, H‐12, H‐6'), 6.60–6.20 (m, 2H, Ar‐H, H‐3', H‐5'), 4.60–4.56 (m, 4H, H‐19, H‐20), 3.71‐3.69 (m, 1H, H‐13), 2.29 (m, 6H, 2‐CH₃), 1.41‐1.38 (m, 3H, H‐15). ¹³C‐NMR (150 MHz, DMSO‐d₆ ): δ 174.4 (C‐14), 169.2 (2C═O), 166.4 (C‐4'), 161.6 (C‐2'), 159.7 (C‐3), 154.5 (C‐4″, C‐4″′), 147.8 (C‐1″, C‐1″′), 146.0 (C‐16), 136.7 (C‐17, C‐18), 136.9 (C‐7), 134.9 (C‐1), 130.7 (C‐5), 129.5 (C‐9, C‐11), 128.7 (C‐6'), 128.6 (C‐8, C‐12), 127.7 (C‐10), 126.4 (C‐4), 124.1 (C‐6), 123.8 (C‐5'), 115.0 (C‐3'), 114.5 (C‐2), 108.6 (C‐2″, C‐2″′), 104.3 (C‐1'), 100.3 (C‐3″, C‐3″′), 67.0 (C‐19, C‐20), 43.5 (C‐13), 18.3 (C‐15), 13.5 (2‐CH₃). HRMS (ESI⁺): [M + NH₄]⁺ calcd for C₃₈H₃₉FN₇O₇: 724.4251; found 724.4246.

3.4.14. 2‐(2‐fluoro‐[1,1'‐biphenyl]‐4‐yl)‐N'‐(4‐(2‐(2‐(2‐hydroxynaphthalen‐1‐yl)methylene)hydrazinyl)‐2‐oxoethyl)‐2‐((2‐(2‐(2‐hydroxynaphthalen‐1‐yl)methylene)hydrazinyl)‐2‐oxoethyl)peroxy)benzylidene)propanehydrazide (17)

Yield: 72%; ¹H‐NMR (600 MHz, DMSO‐d₆ ): δ 9.52 (s, 2H, 2‐OH), 8.84 (s, 1H, H‐16), 8.07 (s, 2H, H‐17, H‐18), 7.86–7.72 (m, 2H, Ar‐H, H‐2, H‐5), 7.64 (t, J = 9.0 Hz, 4H, Ar‐H, H‐6″, H‐7″, H‐6″′, H‐7″′), 7.41 (t, J = 7.2 Hz, 2H, Ar‐H, H‐9, H‐11), 7.50–7.41 (m, 3H, Ar‐H, H‐6, H‐8, H‐12), 7.15 (t, J = 7.2 Hz, 1H, Ar‐H, H‐10), 7.37–7.33 (m, 1H, Ar‐H, H‐6'), 6.91 (d, J = 7.2 Hz, 2H, Ar‐H, H‐8″, H‐8″′), 6.67–6.645 (m, 8H, Ar‐H, H‐3', H‐5', H‐3″, H‐4″, H‐5″, H‐3″′, H‐4″′, H‐5″′), 4.62‐4.45 (m, 4H, H‐19, H‐20), 3.73‐3.71 (m, 1H, H‐13), 1.41 (d, J = 7.2 Hz, 3H, H‐15). ¹³C‐NMR (150 MHz, DMSO‐d₆ ): δ 174.4 (C‐14), 171.0 (C‐2″, C‐2″′), 169.2 (2C═O), 166.4 (C‐4'), 161.6 (C‐2'), 159.7 (C‐3), 146.0 (C‐16), 143.3 (C‐17, C‐18), 136.9 (C‐7), 134.9 (C‐1), 133.3 (C‐4″, C‐4″′), 133.0 (C‐9″, C‐9″′), 130.7 (C‐5), 129.5 (C‐9, C‐11), 129.1 (C‐10″, C‐10″′), 128.7 (C‐6'), 128.6 (C‐8, C‐12), 128.3 (C‐5″, C‐5″′), 127.7 (C‐10), 127.1 (C‐7″, C‐7″′), 126.4 (C‐4), 124.1 (C‐6), 123.8 (C‐5'), 122.4 (C‐6″, C‐6″′), 121.4 (C‐3″, C‐3″′), 118.6 (C‐8″, C‐8″′), 115.0 (C‐3'), 114.5 (C‐2), 108.5 (C‐1″, C‐1″′), 104.3 (C‐1'), 66.9 (C‐19, C‐20), 43.5 (C‐13), 18.3 (C‐15). HRMS (ESI⁺): [M + H]⁺ calcd for C₄₈H₄₃FN₇O₇: 848.4286; found 848.4437.

3.4.15. 2,2'‐((4‐(2‐(2‐(2‐fluoro‐[1,1'‐biphenyl]‐4‐yl)propanoyl)hydrazono)methyl)‐1,3‐phenylene)bis(oxy))bis(N'‐(3‐hydroxy benzylidene)acetohydrazide) (18)

Yield: 70%; ¹H‐NMR (600 MHz, DMSO): δ 10.86 (s, 1H, ─NH), 10.69 (s, 2H, 2‐NH), 8.96 (s, 2H, 2‐OH), 8.39 (s, 2H, H‐19, H‐20), 8.25 (s, 1H, H‐16), 7.50–7.47 (m, 4H, Ar‐H, H‐6', H‐5, H‐8, H‐12), 7.45–7.42 (m, 3H, Ar‐H, H‐6″, H‐6″′, H‐5'), 7.37–7.33 (m, 2H, Ar‐H, H‐9, H‐11), 7.27–7.23 (m, 4H, Ar‐H, H‐2, H‐2″, H‐2″′, H‐3'), 7.10–7.06 (m, 2H, Ar‐H, H‐5″, H‐5″′), 6.98–6.97 (d, J = 9.0 Hz, 2H, Ar‐H, H‐4″, H‐4″′), 6.88 (t, J = 7.9 Hz, 1H, Ar‐H, H‐10), 6.80 (t, J = 8.0 Hz, 1H, Ar‐H, H‐6), 4.65‐4.48 (m, 4H, H‐17, H‐18), 3.80‐3.76 (m, 1H, H‐13), 1.44 (d, J = 7.0 Hz, 3H, H‐15). ¹³C‐NMR (150 MHz, DMSO) δ 174.0 (C‐14), 168.9 (2C═O), 162.7 (C‐3″, C‐3″′), 159.6 (C‐4'), 158.0 (C‐3), 148.4 (C‐16), 147.9 (C‐19, C‐20), 146.9 (C‐2'), 145.8 (C‐1), 140.6 (C‐1″, C‐1″′), 134.8 (C‐7), 130.7 (C‐5″, C‐5″′), 128.7 (C‐9, C‐11), 128.6 (C‐5), 127.8 (C‐8, C‐12), 126.7 (C‐10), 126.3 (C‐4), 123.8 (C‐6), 122.0 (C‐6″, C‐6″′), 120.5 (C‐6'), 119.3 (C‐4″, C‐4″′), 118.3 (C‐2), 115.2 (C‐2″, C‐2″′), 114.9 (C‐5'), 113.7 (C‐3'), 112.8 (C‐1'), 66.4 (C‐17, C‐18), 43.4 (C‐13), 18.1 (C‐15). HRMS (ESI⁺): [M + NH₄]⁺ calcd for C₄₀H₃₉FN₇O₇: 748.2551; found 748.2918.

3.4.16. 2,2'‐((4‐(2‐(2‐(2‐fluoro‐[1,1'‐biphenyl]‐4‐yl)propanoyl)hydrazono)methyl)‐1,3‐phenylene)bis(oxy))bis(N'‐(2‐chloro benzylidene)acetohydrazide) (19)

Yield: 75%; ¹H‐NMR (600 MHz, DMSO): δ 11.63 (s, 1H, ─NH), 11.41 (s, 2H, 2‐NH), 8.93 (s, 1H, H‐16), 8.69 (s, 2H, H‐19, H‐20), 8.18 (s, 1H, Ar‐H, H‐2), 7.92 (s, 1H, Ar‐H, H‐3'), 7.88 (d, J = 8.3 Hz, 2H, Ar‐H, H‐6″, H‐6″′), 7.70–7.66 (m, 3H, Ar‐H, H‐5″, H‐5″′, H‐5), 7.56 (d, J = 8.2 Hz, 2H, Ar‐H, H‐8, H‐12), 7.51–7.46 (m, 4H, Ar‐H, H‐3″, H‐4″, H‐3″′, H‐4″′), 7.44–7.41 (m, 3H, Ar‐H, H‐9, H‐11, H‐6'), 7.38–7.34 (m, 1H, Ar‐H, H‐10), 7.26–7.24 (m, 1H, Ar‐H, H‐6), 6.67 (br.s, 1H, Ar‐H, H‐5'), 4.61 (s, 2H, H‐17), 4.55 (s, 2H, H‐18), 3.77‐3.73 (m, 1H, H‐13), 1.41 (d, J = 7.0 Hz, 3H, H‐15). ¹³C‐NMR (150 MHz, DMSO) δ 174.3 (C‐14), 169.2 (2C═O), 159.6 (C‐4'), 158.0 (C‐3), 149.8 (C‐2'), 144.1 (C‐16), 143.4 (C‐1), 140.3 (C‐19, C‐20), 135.0 (C‐2″, C‐2″′), 134.9 (C‐7), 131.8 (C‐6'), 131.3 (C‐1″, C‐1″′), 130.7 (C‐5), 130.5 (C‐3″, C‐3″′), 128.7 (C‐9, C‐11), 128.6 (C‐4″, C‐4″′), 128.5 (C‐6″, C‐6″′), 127.8 (C‐8, C‐12), 127.7 (C‐5″, C‐5″′), 126.7 (C‐4), 126.3 (C‐10), 123.8 (C‐6), 115.0 (C‐2), 114.8 (C‐3'), 112.3 (C‐1', C‐5'), 66.4 (C‐17, C‐18), 43.4 (C‐13), 18.2 (C‐15). HRMS (ESI⁺): [M + H]⁺ calcd for C₄₀H₃₃Cl₂FN₆O₅: 768.1844; found 770.0941.

3.4.17. 2,2'‐((4‐(2‐(2‐(2‐fluoro‐[1,1'‐biphenyl]‐4‐yl)propanoyl)hydrazono)methyl)‐1,3‐phenylene)bis(oxy))bis(N'‐(thiophen‐2‐yl methylene)acetohydrazide) (20)

Yield: 73%; ¹H‐NMR (600 MHz, DMSO): δ 11.48 (s, 2H, 2‐NH), 11.27 (s, 1H, ─NH), 8.37 (s, 1H, H‐16), 8.05 (s, 2H, H‐19, H‐20), 7.57 (br.s, 2H, Ar‐H, H‐2, H‐3'), 7.47–7.42 (m, 3H, Ar‐H, H‐6, H‐8, H‐12), 7.40–7.37 (m, 4H, Ar‐H, H‐2″, H‐4″, H‐2″′, H‐4″′), 7.34–7.29 (m, 2H, Ar‐H, H‐3″, H‐3″′), 7.22–7.17 (m, 3H, Ar‐H, H‐5, H‐10, H‐5'), 7.06–7.03 (m, 2H, Ar‐H, H‐9, H‐11), 6.63–6.61 (m, 1H, Ar‐H, H‐6'), 4.57 (s, 2H, H‐17), 4.51 (s, 2H, H‐18), 3.69–3.66 (m, 1H, H‐13), 1.38 (d, J = 6.0 Hz, 3H, H‐15). ¹³C‐NMR (150 MHz, DMSO) δ 173.9 (C‐14), 169.0 (2C═O), 159.6 (C‐3), 157.9 (C‐4'), 155.7 (C‐2'), 143.3 (C‐1″, C‐1″′), 142.2 (C‐16), 139.0 (C‐1, C‐7), 137.9 (C‐4), 134.8 (C‐5), 130.8 (C‐2″), 130.6 (C‐2″′), 130.1 (C‐6'), 128.8 (C‐6), 128.6 (C‐8, C‐12), 128.5 (C‐9, C‐11), 128.5 (C‐4″, C‐4″′), 128.3 (C‐5'), 127.7 (C‐19, C‐20), 127.7 (C‐10), 123.9 (C‐3″), 123.7 (C‐3″′), 115.3 (C‐2), 114.9 (C‐3'), 100.1 (C‐1'), 66.4 (C‐17), 62.5 (C‐18), 43.4 (C‐13), 18.1 (C‐15). HRMS (ESI⁺): [M + H]⁺ calcd for C₃₆H₃₁FN₆O₅S₂: 710.1781; found 710.4733.

3.4.18. 2,2'‐((4‐(2‐(2‐(2‐fluoro‐[1,1'‐biphenyl]‐4‐yl)propanoyl)hydrazono)methyl)‐1,3‐phenylene)bis(oxy))bis(N'‐(naphthalen‐1‐ylmethylene)acetohydrazide) (21)

Yield: 64%; ¹H‐NMR (600 MHz, DMSO): δ 11.77 (s, 2H, 2‐NH), 11.50 (s, 1H, ─NH), 8.93 (s, 1H, H‐16), 8.35 (s, 2H, H‐19, H‐20), 8.07 (s, 2H, Ar‐H, H‐2, H‐3'), 7.86 (t, J = 8.2 Hz, 4H, Ar‐H, H‐3″, H‐6″, H‐3″′, H‐6″′), 7.76 (t, J = 8.1 Hz, 2H, Ar‐H, H‐9, H‐11), 7.63 (d, J = 9.0 Hz, 2H, Ar‐H, H‐2″, H‐2″′), 7.49–7.45 (m, 4H, Ar‐H, H‐8″, H‐8″′, H‐8, H‐12), 7.44–7.41 (m, 4H, Ar‐H, H‐4″, H‐5″, H‐4″′, H‐5″′), 7.38–7.34 (m, 2H, Ar‐H, H‐5, H‐10), 7.26–7.23 (m, 4H, Ar‐H, H‐7″, H‐7″′, H‐6, H‐6'), 6.67–6.64 (m, 1H, Ar‐H, H‐5'), 4.44‐4.32 (m, 4H, H‐17, H‐18), 3.75‐3.72 (m, 1H, H‐13), 1.42 (d, J = 6.2 Hz, 3H, H‐15). ¹³C‐NMR (150 MHz, DMSO) δ 174.4 (C‐14), 169.3 (2C═O), 166.4 (C‐4'), 160.6 (C‐2'), 159.6 (C‐3), 145.7 (C‐16), 143.3 (C‐1), 141.8 (C‐19, C‐20), 136.0 (C‐10″, C‐10″′), 134.9 (C‐6'), 134.2 (C‐7), 133.1 (C‐9″, C‐9″′), 132.6 (C‐1″, C‐1″′), 130.6 (C‐5), 130.0 (C‐8″, C‐8″′), 129.1 (C‐10), 128.9 (C‐4″, C‐5″, C‐4″′, C‐5″′), 128.7 (C‐9, C‐11), 128.6 (C‐3″, C‐6″, C‐3″′, C‐6″′), 128.4 (C‐7″, C‐7″′), 127.8 (C‐8, C‐12), 127.7 (C‐2″, C‐2″′), 126.7 (C‐4), 124.1 (C‐6), 123.8 (C‐3'), 115.2 (C‐2), 107.8 (C‐5'), 100.3 (C‐1'), 66.4 (C‐17, C‐18), 43.5 (C‐13), 18.2 (C‐15). HRMS (ESI⁺): [M + H]⁺ calcd for C₄₈H₄₀FN₆O₅: 799.2966; found 799.8718.

3.4.19. 2,2'‐((4‐((2‐(2‐(2‐fluoro‐[1,1'‐biphenyl]‐4‐yl)propanoyl)hydrazono)methyl)‐1,3‐phenylene)bis(oxy))bis(N'‐(butylidene) acetohydrazide) (22)

Yield: 58%; ¹H‐NMR (600 MHz, DMSO‐d₆ ): δ 11.51 (br.s, 1H, ─NH), 9.36 (s, 2H, 2‐NH), 8.30 (s, 1H, H‐16), 8.75 (br.s, 2H, H‐17, H‐18), 7.49–7.40 (m, 5H, Ar‐H, H‐8‐12), 7.39–7‐34 (m, 1H, Ar‐H, H‐5), 7.29–7.21 (m, 2H, Ar‐H, H‐6, H‐6'), 7.17–7.15 (m, 1H, Ar‐H, H‐2), 7.67–7.51 (m, 2H, Ar‐H, H‐3', H‐6'), 4.67‐4.52 (m, 4H, H‐19, H‐20), 3.63‐3.60 (m, 1H, H‐13), 2.13‐2.11 (m, 4H, H‐1″, H‐1″′), 1.48‐1.34 (m, 4H, H‐2″, H‐2″′), 1.26‐1.19 (m, 3H, H‐15), 0.90‐0.76 (m, 6H, H‐3″, H‐3″′). ¹³C‐NMR (150 MHz, DMSO‐d₆ ): δ 173.8 (C‐14), 168.6 (2C═O), 163.3 (C‐4'), 159.6 (C‐3), 151.2 (C‐17, C‐18), 147.2 (C‐16), 143.8 (C‐2'), 135.0 (C‐1), 134.9 (C‐7), 130.7 (C‐5), 130.5 (C‐6'), 128.7 (C‐9, C‐11), 128.6 (C‐8, C‐12), 127.8 (C‐10), 126.6 (C‐4), 126.3 (C‐1'), 124.1 (C‐6), 123.8 (C‐5'), 115.2 (C‐3'), 114.8 (C‐2), 66.2 (C‐19, C‐20), 43.3 (C‐13), 33.8 (C‐1″, C‐1″′), 19.4 (C‐2″, C‐2″′), 18.2 (C‐15), 13.7 (C‐3″, C‐3″′). HRMS (ESI⁺): [M + H]⁺ calcd for C₃₄H₃₉FN₆O₅: 630.2966; found 639.1949.

3.4.20. 2,2'‐((4‐(2‐(2‐(2‐fluoro‐[1,1'‐biphenyl]‐4‐yl)propanoyl)hydrazono)methyl)‐1,3‐phenylene)bis(oxy))bis(N'‐(hexylidene) acetohydrazide) (23)

Yield: 55%; ¹H‐NMR (600 MHz, DMSO‐d₆ ): δ 11.53 (br.s, 2H, 2‐NH), 11.22 (br.s, 1H, ─NH), 8.62 (br.s, 1H, H‐16), 8.36‐8.33 (br.s, 1H, Ar‐H, H‐2), 7.78 (br.s, 2H, H‐17, H‐18), 7.60 (br.s, 1H, Ar‐H, H‐10), 7.51–7‐37 (m, 2H, Ar‐H, H‐8, H‐12), 7.32–7.24 (m, 2H, Ar‐H, H‐9, H‐11), 6.63–6.53 (m, 5H, Ar‐H, H‐5, H‐6, H‐3', H‐5', H‐6'), 4.65‐4.50 (m, 4H, H‐19, H‐20), 3.63‐3.60 (m, 1H, H‐13), 2.18 (br.s, 4H, H‐1″, H‐1″′), 1.73 (m, 8H, H‐2″, H‐3″, H‐2″′, H‐3″′), 1.44‐1.41 (m, 4H, H‐4″, H‐4″′), 1.27‐1.22 (m, 3H, H‐15), 0.84‐0.70 (m, 6H, H‐5″, H‐5″′). ¹³C‐NMR (150 MHz, DMSO‐d₆ ): δ 173.8 (C‐14), 168.6 (2C═O), 163.3 (C‐4'), 159.6 (C‐3), 154.1 (C‐17, C‐18), 146.0 (C‐16), 143.8 (C‐2'), 135.0 (C‐1), 134.9 (C‐7), 130.7 (C‐5), 130.5 (C‐6'), 128.7 (C‐9, C‐11), 128.6 (C‐8, C‐12), 127.8 (C‐10), 126.6 (C‐4), 126.3 (C‐1'), 124.1 (C‐6), 123.8 (C‐5'), 115.2 (C‐3'), 114.8 (C‐2), 69.0 (C‐19, C‐20), 43.3 (C‐13), 30.8 (C‐3″, C‐3″′), 25.5 (C‐1″, C‐1″′), 24.3 (C‐2″, C‐2″′), 21.9 (C‐4″, C‐4″′), 18.2 (C‐15), 14.7 (C‐5″, C‐5″′). HRMS (ESI⁺): [M + H]⁺ calcd for C₃₈H₄₈FN₆O₅: 688.3592; found: 688.6572.

3.4.21. 2,2'‐((4‐(2‐(2‐(2‐fluoro‐[1,1'‐biphenyl]‐4‐yl)propanoyl)hydrazono)methyl)‐1,3‐phenylene)bis(oxy))bis(N'‐(3‐bromo benzylidene)acetohydrazide) (24)

Yield: 76%; ¹H‐NMR (600 MHz, DMSO): δ 11.77 (s, 2H, 2‐NH), 11.54 (s, 1H, ─NH), 8.77 (s, 1H, H‐16), 8.24 (s, 1H, H‐19), 8.31 (br.s, 1H, H‐20), 8.00–7.92 (m, 3H, Ar‐H, H‐6, H‐8, H‐12), 7.75 (d, J = 7.7 Hz, 2H, Ar‐H, H‐6″, H‐6″′), 7.66 (t, J = 7.6 Hz, 2H, Ar‐H, H‐5″, H‐5″′), 7.59‐7.51 (m, 5H, Ar‐H, H‐2″, H‐2″′, H‐6', H‐4″, H‐4″′), 7.47–7.44 (m, 4H, Ar‐H, H‐2, H‐5, H‐10, H‐5'), 7.34 (t, J = 8.2 Hz, 2H, Ar‐H, H‐9, H‐11), 6.79–6.74 (m, 1H, Ar‐H, H‐3'), 4.85‐4.80 (m, 4H, H‐17, H‐18), 3.87‐3.83 (m, 1H, H‐13), 1.52 (d, J = 6.0 Hz, 3H, H‐15). ¹³C NMR (150 MHz, DMSO) δ 174.1 (C‐14), 168.9 (2C═O), 166.3 (C‐4'), 159.6 (C‐3), 158.0 (C‐2'), 149.0 (C‐16), 147.1 (C‐19, C‐20), 134.8 (C‐1, C‐7), 130.7 (C‐4″, C‐4″′), 128.7 (C‐6″, C‐6″′), 128.6 (C‐9, C‐11), 128.6 (C‐5″, C‐5″′), 127.8 (C‐6'), 127.7 (C‐8, C‐12), 126.9 (C‐10), 126.7 (C‐4), 126.2 (C‐5), 123.8 (C‐3″, C‐3″′), 121.8 (C‐2″, C‐2″′), 121.3 (C‐6), 115.2 (C‐3'), 114.9 (C‐2), 111.4 (C‐1″, C‐1″′), 108.2 (C‐5'), 107.9 (C‐1'), 66.4 (C‐17, C‐18), 43.4 (C‐13), 18.5 (C‐15). HRMS (ESI⁺): [M + H]⁺ calcd for C₄₀H₃₃Br₂FN₆O₅: 856.5474; found: 857.4328.

3.5. In Vitro Tyrosinase Inhibitory Assay

The tyrosinase inhibitory activity of the product compounds was carried out using an already published method in the literature [38].

3.6. Quantum Chemical Calculations

To examine the chemical reactivity as well as electronic properties of the synthesised inhibitors, we performed DFT calculations using the Gaussian 16 software package [39].

3.7. Geometry Optimisation and Reactivity Indices

The ground‐state geometries of all the synthesised tris‐Schiff bases were optimised fully in the gas phase without imposing any symmetry constraints. The calculations were performed using the long‐range corrected CAM‐B3LYP functional, which is well‐suited for describing charge‐transfer systems, and Grimme's D3 empirical dispersion correction method was used to account for van der Waals interactions [40]. All atoms were calculated using the minimal split‐valence (MSV). The frequency calculations were carried out at the same theoretical level to confirm that the optimised structures indeed represent a minimum energy point (no imaginary frequencies). The energy values of the HOMO and LUMO were extracted from the optimised structure. These energies were used to calculate a series of global reactivity indices based on conceptual DFT, like the global electrophilicity index (ω = χ ²/2η), electronegativity (χ = −(E_HOMO + E_LUMO)/2), chemical softness (σ = 1/η), chemical hardness (η = (E_LUMO − E_HOMO)/2) and the energy gap (E _g = LUMO − HOMO). The MEP maps were considered on the electron density surface (isovalue = 0.002 a.u.) of the DFT‐optimised geometries. The MEP maps imagine the charge distribution, with potential values reaching from red (negative potential, electron‐rich regions) to blue (positive potential, electron‐poor regions) that provide understanding into the sites for nucleophilic as well as electrophilic interactions.

3.8. Molecular Docking

The 3D crystal structure data of mushroom tyrosinase were obtained from the Protein Data Bank (PDB ID: 2Y9X). The protein structure was pre‐processed using the CASTpFold module that included removing co‐crystallised ligands and water molecules, adding hydrogen atoms, determining bond types and constructing disulfide bonds. The 2D structures of all inhibitors, including the reference compound kojic acid, were converted to 3D structures. Subsequently, the ligand was pre‐processed using appropriate software to ensure it was in the correct tautomeric and ionisation state at pH 7.4 ± 0.5, and conformational search and energy minimisation calculations were performed. Finally, molecular docking simulations were carried out using AutoDock Vina software [41]. We defined a grid space with dimensions of 60 × 60 × 60 Å and a grid spacing of 1.0 Å, centring it on the active site to encompass the two catalytic copper ions and the surrounding key residues. To thoroughly explore the conformational space, the search depth was set to 20. For each ligand, 10 possible binding conformations were generated and ranked based on their binding energy (kcal/mol). The conformation with the lowest (most favourable) binding energy was selected for further analysis and visualisation.

3.9. Molecular Dynamics Simulation

To explore the collective dynamics and intrinsic flexibility of the tyrosinase‐compound 5 complex, NMA was performed using the iMODS module [42]. The lowest‐scoring docked conformation of 5 complexed with tyrosinase (PDB: 2Y9X) was used as the input structure. iMODS calculates collective motions based on an internal coordinate mechanics approach. These modules provided detailed analyses of the protein's dynamics, including deformability, B‐factors, eigenvalues, variance associated with each normal mode and a dynamic cross‐correlation matrix (DCCM) to investigate the coupled motions between protein residues.

4. Conclusion

The current study is based on the synthesis of novel tris‐Schiff base derivatives of marketed accessible flurbiprofen in better yields through multi‐step reactions. Structures of these compounds were deduced through HR‐ESI‐MS, ¹³C‐ and ¹H‐NMR spectroscopic methods and finally screened for their in vitro tyrosinase inhibitory potential. All the synthetic derivatives attributed noteworthy inhibition except compounds (11, 14 and 16), which were found inactive. However, eight compounds (5, 6, 7, 8, 19, 24, 22 and 18) in the series were found potent inhibitory effect with IC₅₀ values ranging from IC₅₀ of 8.7 ± 0.4 µM to 18.9 ± 0.5 µM, compared with the standard kojic acid (IC₅₀ = 18.7 ± 0.5 µM). Furthermore, the remaining nine compounds showed good to less inhibitory activity. Molecular docking exposed strong hydrogen bonding and optimal binding geometry, while MD and NMA confirmed reduced conformational flexibility and active site stabilisation. The integrated computational studies suggested that compound 5 inhibits tyrosinase by forming a stable complex within the active site, leading to a global reduction in enzyme flexibility. The robust association among docking scores and experimental IC₅₀ values further authorises its inhibitory mechanism of action via stable active‐site engagement. The exact kinetic mode of inhibition (e.g., competitive or non‐competitive) remains a subject for future investigation. General, compound 5 presents an auspicious framework for the rational design of next‐generation anti‐melanogenic agents.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File 1: cbdv71621‐sup‐0001‐SuppMat.docx.

Data Availability Statement

Data of the compounds are available in the Supporting Information.