Thiadiazole Functionalized Salicylaldehyde-Schiff Base as a pH-responsive and chemo-reversible “Turn-Off” fluorescent probe for selective cu (II) detection: Logic Gate Behaviour and Molecular Docking Studies

A novel thiadiazole functionalized schiff base chemoreceptor (E)-2,4-dichloro-6-(((5-mercapto-1,3,4-thiadiazol-2-yl)imino)methyl)phenol (SB-1) has been synthesized and characterized spectroscopically by using various techniques. Its photophysical behaviour was scanned towards a variety of metal ions in mixed aqueous media. The chemosensor (SB-1) displayed excellent selectivity towards Cu2+ ion through fluorescent diminishment (turn-off phenomenon). Colorimetric analyses showed a rapid colour change from yellow to dark red under visible light upon addition of Cu2+ ions. Interestingly, the original yellow colour reappeared back instantly after the addition of EDTA2− anions, thus confirming the reversible nature of SB-1. Competitive experiments validated no interference from the other co-existing metal ions in the recognition process of SB-1 towards Cu2+ ion. Job’s plot confirmed 1:1 binding stoichiometry between SB-1 and Cu2+ ion with the binding constant value of 3.87 × 104 M− 1. The limit of detection was determined to be 1.01 × 10− 7 M suggesting good sensitivity of SB-1 towards Cu2+ ions. Furthermore, pH-dependent UV-Vis spectral behaviour of SB-1 confirmed that it could act as an effective optical pH-sensor for highly acidic environment as well. Portable nature of probe SB-1 was explored by fabricating “easy-to-use” paper test strips, which allow robust and rapid detection of Cu2+ ions. Based on the multi-responsive properties of SB-1, a ‘NOR’ logic gate was constructed by applying Cu2+ and EDTA2− as chemical inputs (ln1: Cu2+, ln2: EDTA2−) while emission intensity observed at 560 nm was considered as output signal (O1). DFT optimized geometries confirmed that chemosensor SB-1 exists in Azo form (Enol form) in its ground state. Molecular docking of the SB-1 and its copper complex, into the binding site of TRK protein tyrosine kinase (PDB: 1t46) was also carried out to explore their biological activity and their potential use as TRK inhibitors.

functions of living organisms and absorbed in the form of metal ions as micro and macronutrients which are essential for growth and development [2]. The uptake of metal ions within the prescribed limit is vital for the smooth functioning and maintenance of life. However, over accumulation of these ions could lead to serious health concerns, growth disorders, severe malfunctions, carcinogenesis or even death [3]. Therefore, developing effective procedures for qualitative and quantitative identification of bio-accumulative metal ions is of utmost significance.
Copper is the third most abundant essential transition metal ion present in the human body. It offers multifarious utilization in everyday lives attributed to its versatile structural and physical characteristics [4]. It is extensively exploited in almost all modern electronics, telecommunication, medical/surgical equipment, jewellery, plumbing as well as in numerous household items [5]. Besides, it also plays imperative role in cellular physiology [6]. It functions as a cofactor and/or as integral component for a number of metalloenzymes and actively participates in numerous physiologic processes such as respiration, tissue maturation, antioxidative defense, energy metabolism, neural transmission, and iron metabolism [7]. Copper accumulated into human body through air breathing, copper containing compounds, and drinking water. The intracellular concentration of copper ions must be kept at an appropriate level in order to establish cellular processes. As per WHO parameters, the recommended daily intake of copper ions for an adult human is approximated to be 1-3 mg [8]. The excessive intake from this limit is deleterious to human health and may lead to nausea, irritation of nose, vomiting, and diarrhoea [9]. Moreover, the unregulated dosage of copper can cause oxidative stress and disorder associated with neurodegenerative diseases including Wilson, Menkei, familiar amyotrophic lateral sclerosis, and Alzheimer [10]. Both chronically and acutely, the free copper is also detrimental for aquatic life. Therefore, it is crucial to detect copper ions selectively and sensitively to control its impact on human health and environment.
Over the years, a number of analytical techniques were developed and employed for the detection of metal ions. These includes atomic absorption spectroscopy (AAS) [11][12][13], ion chromatography (IC) [14,15], inductively coupled plasma mass spectrometry (ICP-MS) [16,17], thermometric titration method [18], pH electrode [19] etc. However, aforementioned techniques suffer from one and/or more drawbacks such as tedious procedures for sample preparation, sophisticated instrumentation, requirement of skilled manpower and lack of achievability and practicability. Hence, there is a pressing need to establish alternative approaches with improved properties for the rapid and robust detection of toxic metal ions.
Design, synthesis and utilization of low molecular weight fluorescent probes (LMFPs) targeting selective detection of biologically or industrially relevant metal ions have been receiving consistent recognition over the last several years [20]. These chemo sensors work on the principle of switching fluorescent emission either 'ON' or 'OFF' mechanism and have proven to be so advantageous over classical techniques owing to their easy synthesis, cost-effectiveness, excellent sensitivity, real-time response, and target ion-induced naked eye visualization. Among the various LMFPs explored, Schiff bases (azomethines) are considered to be the privileged ligand for fluorescent sensing due to their excellent chelating tendency, exemplary chromogenic properties, and structural flexibility [21]. These compounds are easy to prepare in good to high yields under short reaction time while necessitating only a minimum of effort. In particular, Schiff base compounds derived from the bioactive heterocyclic amines, which contain a strong fluorophoric unit, are being widely implemented as potential fluorescent sensing probes.
2-amino-1,3,4-thiadiazoles are by far the most important class of five-membered heterocycles reported to have remarkable physiochemical properties i.e. crystal polymorphism, solvatomorphism [22][23][24][25], and dual fluorescence emission [26]. Thiadiazoles comprise of a key "thiazole" chromophoric core, and therefore are intrinsically fluorescent [27,28]. Incorporation of 2-amino-1,3,4-thiadiazoles into the backbone of target schiff base compounds, therefore, enhance their colorant and chromophoric strength, and consequently make them very desirable as colorimetric/fluorescent detectors. Furthermore, the insertion of structurally specific aldehydic moieties, such as salicylaldehyde derivatives, provide much better metal-binding ability to target azomethines and in turn, induce substantial changes in their photophysical and/or colorimetric properties. The superior metal-coordinating ability of salicylaldehyde motif is primarily due to the presence of hydroxyl group (-OH) adjacent to imine group (> C = N), which actively participates in the complexation through oxygen atom. Henceforth, it can be concluded from the above-mentioned facts that incorporation of these two privileged scaffolds in a single entity must be of considerable importance, and would definitely ameliorate the performance of the chemosensor. Moreover, Schiff bases are also found to be excellent pharmcophore, and therefore find promising applications in modern drug discovery [29,30]. A variety of schiff base compounds has been reported to exhibit a broad spectrum of biological activities, including anti-tumour, anti-bacterial, antifungicidal and anticarcinogenic properties [31,32] which also tempted us to explore the role of our synthesized compound as an anticancer drug by attempting in-silico docking studies on target protein.
Therefore, in continuation to our ongoing research interest aiming towards the development of reasonably low cost and efficient fluorescent-colorimetric probes [33][34][35], we have designed and synthesized a Schiff base chemoreceptor (SB-1) by employing 5-amino-1,3,4-thiadiazole-2(3H)-thione and 3,5-dichlorosalicylaldehyde as reaction precursors. The probe displays selective fluorescence "turn-off" behaviour upon coordinating with Cu 2+ ions and successfully allows "naked-eye' detection of Cu 2+ ions by inducing instant visible colour change from yellow to dark red in the corresponding solution. The pH-responsive behaviour of Schiff base further enabled us to explore its role as an optical pH-sensor as well. We explored the practical applicability of by constructing molecular logic gates and sensing paper strips. We explored a potential biological use of Schiff base as an anti-cancer drug molecule by conducting in-silico docking on target protein. Concentrating on anticarcinogenic properties, in this paper, molecular docking of synthesized schiff base chemoreceptor SB-1 and its Cu complex was carried out to see if these compounds can be used as anticancer drug molecules for target receptor protein. We chose to test this with tyrosine kinase enzymes as the receptor. Tyrosine kinase enzymes are a part of many cell functions, including cell signalling, growth, division, and have been found at high levels in many types of cancer cells [36]. Tyrosine kinase inhibitors, therefore, hold much importance in cancer treatments [37].

Synthesis and characterization of SB-1
The desired probe SB-1 was synthesized using the protocol illustrated in Scheme 1. Ethanolic solutions of 2,5-dichlorosalicylaldehyde (1) and 5-amino-1,3,4-thiadiazole-2-thiol (2) were mixed with constant stirring together in a 100 mL round-bottom flask followed by the addition of 2-3 drops of acetic acid. The resulting mixture was refluxed for one hour. The completion of reaction was observed by TLC using ethylacetate : petroleum ether (50 : 50) as eluent. Deep yellow precipitate were filtered off and washed several times with cold water. The crude product was then purified by recrystallization from hot ethanol and dried over P 4 O 10 to give the pure product SB-1 as dark red solid. The product SB-1 was characterized spectroscopically by IR, 1 H and 13 C NMR, and by mass spectra.

Receptor-spacer-fluorophore paradigm for chemo sensor SB-1:
The "Receptor-Spacer-Fluorophore" paradigm for chemo sensor SB-1 is illustrated in Fig. 1. The construction of compound SB-1 involves the assembling of a salicylaldehyde 'receptor' unit (primarily responsible for the selective analyte binding) coupled to a thiadiazole 'fluorophore' subunit (which generate spectral/optical response due to receptoranalyte interaction) through an imine (-C = N) spacer. The role of spacer is to modulate the electronic interactions between receptor and fluorophore.

UV-Vis spectral analysis
The UV-Vis absorption spectrum of probe SB-1 showed two absorption maxima, one in the shorter wavelength region centred at 298 nm and a broad band in the longer wavelength region ranging from 434 − 439 nm (λ max = 437 nm) (Fig. 2a). The absorption spectrum remained unaltered upon addition of one equivalent of various metal ions such as Co 2+ , Cd 2+ , Mn 2+ , Hg 2+ , Pb 2+ , Zn 2+ , Sn 2+ , Ni 2+ , Sr 2+ , Mg 2+ , Cr 3+ , Al 3+ , Fe 2+ metal ions (1 × 10 −3 M) to the solution of probe SB-1 (1 × 10 −5 M). However, addition of one equivalent of Cu 2+ ion resulted in the significant changes in the absorption spectrum of SB-1 at long wavelength region. The broad band appeared in the range of 434 − 439 nm underwent hypochromic shift (significant reduction in absorption intensity) along with the slight bathochromic shift (~ 11 nm) of absorption maximum (λ max shifted to 448 nm) (Fig. 2a). However, the absorption maxima centered at 298 nm in shorter wavelength region remain unshifted with the addition of Cu 2+ ions. Thereafter, the absorption spectrum of SB-1 was investigated with varying concentrations of Cu 2+ ions (0-1 equiv.). The intensity of absorption maxima in the region 434 − 439 nm showed gradual diminishment with increasing concentration of Cu 2+ ions (Fig. 2b). However, absorption intensity remained nearly constant and did not change after addition of 1 equiv. of Cu 2+ ions which primarily suggest 1:1 binding mode between SB-1 and Cu 2+ ions.

Effect of water percentage on fluorometric properties of SB-1
The chemo sensor SB-1 exhibited strong emission maximum in 100% methanolic solution (When no water is added). The emission intensity remained nearly constant on increasing the water fraction (f w ) up to 40-50% (MeOH:H 2 O, v/v = 1:1), which clearly nullifies the possibility of "Aggregation-Induced Emission Enhancement (AIEE) Phenomenon". Further increase in dilution (f w > 50%) resulted in the precipitation of SB-1 from the solution due to its poor solubility in high water concentrations which may substantially affect the overall performance of the probe, and could intricately alter the measurements as well. Therefore, MeOH:H 2 O ratio kept at 1:1 during all the photophysical experiments.

Effect of metal ions on photophysical properties of SB-1
The photophysical properties of probe SB-1 has been investigated by determining the changes in UV-Vis absorption and emission spectra of SB-1 (1 × 10 − 5 M) with the addition of different competitive metal ions in MeOH:H 2 O (v/v = 1:1) containing HEPES buffer (1 × 10 − 5 M, pH = 7.2) at room temperature.

Colorimetric studies
Development of colorimetric sensing probes is advantageous as they allow rapid on-site detection of metal ions through visual inspection. Therefore, spectral changes observed in the UV-Vis absorption and emission behaviour of SB-1 upon addition of various metal ions were also inspected by carrying out the colorimetric experiments. Addition of various metal ions to the solution of SB-1 (1 × 10 − 5 M) induced no visible colour change except Cu 2+ ions. A prominent colour change was observed from original yellow to dark red in the presence of Cu 2+ ions (Fig. 4). Therefore, SB-1 could be used as a potential colorimetric chemo sensor for the detection of Cu 2+ ions.

Selective behaviour of SB-1 towards Cu 2+ ions
The selectivity of probe SB-1 towards Cu 2+ ions over other metal ions was further validated by conducting the competitive experiments. The changes in fluorescence intensity of (SB-1 + Cu 2+ ) complex in the presence of various interfering metal ions were investigated. It was observed that emission
Furthermore, the fluorescence titration curve of SB-1 was plotted with gradual addition of varying concentration of Cu 2+ ions (0-1 equiv). The emission intensity of probe SB-1 showed diminishment with incremental amounts of Cu 2+ ions (Fig. 3b). However, emission intensity did not change after the addition of 1 equiv. of Cu 2+ ions indicating 1:1 binding mode between SB-1 and Cu 2+ ions. This was further confirmed by the method of continuous variation.  complex. This fluorometric change was also observed by the change in colour of solution from dark red to original yellow. Subsequent addition of Cu 2+ ion to the solution of (SB-1 + Cu 2+ + EDTA) diminish intensity again along with the colour change from yellow to dark red, thus confirming reversible nature of SB-1. This complexation-decomplexation mechanism has been illustrated in Scheme 2. Therefore, (SB-1)-Cu 2+ complex could also be employed as a secondary sensor for the instant detection of EDTA anions through metal displacement approach. More interestingly, this switching phenomenon could be repeated several times by alternate Cu 2+ /EDTA additions without significant loss of any emission intensity (Fig. 6b).

Binding stoichiometry
The binding stoichiometry of probe SB-1 with Cu 2+ ions was calculated using method of continuous variation i.e. Job's plot (Fig. 7). Job's plot was drawn on the basis of fluorescence titration measurement by altering the mole fraction of Cu 2+ ions while total concentration of the resulting solution kept at constant value. The plot exhibited a maximum when the mole fraction of Cu 2+ was 0.5, which strongly indicates 1:1 complex formation between probe SB-1 and Cu 2+ ions.

Determination of binding constant
Binding constant measures how effectively a ligand complexes with the metal ion. The binding constant (K a ) was evaluated from the fluorescence titration data by using Benesi-Hildebrand Eqs. [38,39]. A graph was plotted between intensity of probe SB-1 in the presence of 10 equiv. of Cu 2+ ions followed by the addition of 10 equiv. of other coexisting metal ions (Co 2+ , Cd 2+ , Mn 2+ , Hg 2+ , Pb 2+ , Zn 2+ , Sn 2+ , Ni 2+ , Sr 2+ , Mg 2+ ,Cr 3+ , Al 3+ , Fe 2+ ) remain as such. Therefore, these metal ions did not produce any considerable interference in the detection of Cu 2+ ions confirming the excellent selectivity of SB-1 towards Cu 2+ ions. These results are compiled in the bar diagram showed in Fig. 5.

Interference of various anions
Practical applicability of probe SB-1 was investigated by monitoring the effect of various anions on the emission spectra of (SB-1)-Cu 2+ complex. It can be depicted from

Limit of detection
The limit of detection of probe SB-1 towards Cu 2+ was calculated from fluorescence titration measurements. A curve of fluorescence intensity was plotted against [Cu 2+ ] at 560 nm. The plot showed linear dependence and the detection limit, point at which curve cuts the ordinate axis, was found to be 1.01 × 10 − 7 M (Fig. 9). This value of limit of detection validates good sensitivity of probe SB-1 towards Cu 2+ ions and falls well within in the range of tolerance limit of Cu 2+ in drinking water. complex was found to be 3.87 × 10 4 M − 1 from the ratio of intercept/slope (linearly dependent coefficient R 2 = 0.988) (Fig. 8). The high value of binding constant indicates strong complex formation between probe SB-1 and Cu 2+ ions.

FT-IR spectral behavior
IR spectrum of free probe SB-1 exhibited characteristic broad band at 3224.98 cm − 1 due to hydroxyl (-OH) stretch, and the band at 1602 cm − 1 corresponds to azomethine group (-C = N) (Fig. 10a). Upon binding with Cu 2+ ions, the broad band disappeared completely whereas azomethine band shifted towards lower frequency region at 1572 cm − 1 (Fig. 10b). These noticeable changes observed in the IR spectrum of SB-1 primarily indicate the involvement of -OH group through phenolic oxygen atom, and azomethine group through nitrogen atom.

H-NMR spectra of SB-in presence of Cu 2+ ion
Binding sites of probe SB-1 involved in the coordination sites with Cu 2+ ions was further confirmed by the changes observed in 1 H-NMR spectra in CDCl 3 /DMSO. Significant changes/shifts were observed in the peak positions after addition of Cu 2+ ions. The broad singlet for phenolic -OH proton at 14.17 ppm in free probe SB-1 disappeared completely upon binding with Cu 2+ ions, which validate the coordination between the Cu and oxygen atom ( Fig. 11a and  b). The azomethine proton appeared as a singlet at 8.53 ppm

Determination of binding sites
The binding ratio of SB-1 and Cu 2+ has already been estimated using Job's plot. Furthermore, the coordinating sites of SB-1 were determined by analyzing the changes observed in IR and 1 H NMR spectra of SB-1 after the addition of Cu 2+ ions.

Effect of pH on absorbance characteristics of SB-1
Effect of pH on the UV-Vis absorption spectrum of SB-1 was investigated in MeOH:H 2 O (1:1, v/v). The solutions of different pH from 1 to 13 were prepared by adding appropriate amount of aq. HCl and aq. NaOH in the solution of probe SB-1. Absorption spectrum of SB-1 exhibited two bands, one centered at 298 nm, and another in the range form 434 − 439 nm. Almost No changes were observed when pH of the solution was shifted to basic region from 7 to 13 (Fig. 12).
Shifting of pH in acidic region from 7 to 2 resulted in the significant diminishment of absorption intensity of band at 298 nm. However, no changes were observed in the colour of the solution of probe SB-1. Additionally, when PH was shifted to downfield region and appeared at 10.09 ppm. This is typically due to the transfer of electron density from the azomethine nitrogen to copper metal clearly indicating the

Sensing experiments with Paper Strips
Rapid development of sensing paper strips is beneficial due to their portability, easy use, and availability. These strips provide instant on-site detection of a particular analyte and therefore negate the dependency on expensive and sophisticated instrumental techniques. Therefore, sensing behaviour of probe SB-1 were monitored using paper test strips. Whatman filter papers were dipped in 1 × 10 − 3 M (MeOH:H 2 O (1:1, v/v)) solution of probe SB-1 for 10 min and then dried in air. The yellow colour of probe SB-1 immersed in test strips (Fig. 13a). The yellow-coloured test strip loaded with SB-1 was converted to dark red instantaneously when dipped in Cu 2+ ion solution (1 × 10 − 3 M, MeOH:H 2 O (1:1, further lowered up to 1, the solution turned to colourless from original yellow which might be due to the existence of tautomeric form II i.e. hydrazone form (Keto form) of probe SB-1 (Scheme 3). These colorimetric responses of probe SB-1 suggested that it could also act as a potential optical pH-sensor for selective detection of H + ions in highly acidic medium when pH < 2.
A plausible mechanism based on the "Intramolecular Charge Transfer (ICT)" process has been illustrated in Scheme 4 for "turn-off" sensing of chemo sensor SB-1 with copper ions. The strong ground state fluorescence of SB-1 is probably due to the intramolecular charge transfer from salicylaldehyde moiety to fluorophoric thiadiazole moiety. Upon chelation with copper ions, ICT ability of phenolic hydroxyl restricted resulting in the diminishment of emission intensity with a blue shift. colorimetric responses of probe SB-1 prompted us to mimic a universal 'NOR' logic gate. A 'NOR' gate could be understood as a combination of two separate logic gates 'OR' and 'NOT'. It is also known as 'Negated OR gate' due to negation of results of OR gate by a NOT function. The presence and absence of chemical inputs (In1 = Cu 2+ and In2 = EDTA 2 ) were assigned with binary digits '1' (ON-state) and '0' (OFF-state), respectively. The output signals were observed at 560 nm. Higher emission intensity of SB-1 was assigned as 1 (ON-state) and the low intensity as 0 (OFFstate). When neither input was present (In1 = In2 = 0), emission intensity of SB-1 at 560 nm was high indicating 'ON' state. Addition of Cu 2+ (In1 = 1, In2 = 0) quenched the emission intensity significantly indicating 'OFF' state. Presence of EDTA 2alone (In1 = 0, In2 = 1) did not alter the emission intensity of SB-1 indicating 'ON' state. Finally, when both the inputs were present (In1 = In2 = 0), system was present in 'ON' state. All these combinations construct a 'truth table' shown in Fig. 14b which finally led to formation of a 'NOR' logic gate (Fig. 14c).

HOMO-LUMO analysis
The optimized structure of SB-1 and its tautomeric form (II, Keto/hydrazone form) was investigated by quantum chemical calculations by DFT using the B3LYP/6311G basis set and represented in Fig. 15. HOMO-LUMO analysis of SB-1 (I, Enol/Azo form) revealed that HOMO is primarily confined over the salicylaldehyde moiety while LUMO is localized over thethiadiazole ring and azo linkage. Therefore, charge transfer occurs from thethiadiazole ring to the salicylaldehyde moiety with an energy separation (ΔE) of v/v)) (Fig. 13b). The original yellow colour of test strip was reappeared back when dark red strip immersed in 1mM EDTA solution (Fig. 13c).

Application as Logic Gate Circuit
Ion-induced logic gate circuits possess remarkable applications in electronics and nano-molecular devices. In present work, we report a simple 'NOR' logic gate at sub micromolar level. The yellow solution of SB-1 changed to dark red upon addition of Cu 2+ ions and the original yellow colour reappeared when EDTA 2ions was added to SB-1 + Cu 2+ solution. Moreover, EDTA 2ions alone as chemical input did not induce any colour change in solution of SB-1. These

Molecular docking analysis:
The binding modes of SB-1 and (SB-1)-Cu to TRK are shown in Figs. 16 and 17. Analysis of the close contacts in the structure of the complex predicted by Vina revealed that SB-1 forms two hydrogen bonds with the target protein. As illustrated from the Fig. 16(c) the hydrogen-bond interaction is with Thr670 and Cys673. In addition, the test compound exhibited additional stabilization through hydrophobic and Van der Waals interactions with nearby amino acid residues, Leu644 and Val654. The docking results reveal that compound SB-1 fit in the groove region with the minimum binding affinity of -8.0 kcal/mol. -3.4406 eV. On the other side, the HOMO-LUMO analysis of Keto form (II) implies that charge transfer occurs between the salicylaldehyde and thiadiazole rings with an energy separation of -3.0866 eV (Fig. 15). Therefore, it can be concluded that more electron density is available in Enol form (Azo form) for binding with Cu 2+ ions due to the possibility of less charge transfer between LUMO and HOMO. In Keto form (II), due to the low energy gap (ΔE = -3.4406 eV), there is more possibility of charge transfer resulting in the reduction of electron density available for Cu 2+ binding. Therefore, it is clear from the above studies that chemo sensor SB-1 exists in its tautomeric form I (Enol/Azo form) in its ground state for metal ion binding. SB-1 complexed in equimolar ratio with Cu 2+ , and exhibited excellent binding ability and sensitivity towards Cu 2+ ions. pH-dependent absorption studies further revealed its colour tuneable properties in acidic range at pH > 2. Portable sensing test strips were fabricated which permit convenient and rapid sensing of copper ions. Based on these characteristics, a 'NOR' logic gate was also constructed. The synthesized

Conclusions
In conclusion, we have designed and synthesized a novel Schiff base (SB-1) for the selective recognition of copper ion through fluorescent "turn-off" mechanism. Reversible studies confirmed that (SB-1)-Cu 2+ complex could also act as a secondary sensor for EDTA anion recognition. Probe compound and its complex with Cu were investigated for the binding affinity to tyrosine kinases receptor (PDB1t46). AutoDockVina 1.2.0 evaluated the binding free energies of this inhibitor into the target c-kit kinase receptor. The docking results indicated that the test compounds have shown

Sensing experiments
While examining the sensing behaviour of SB-1 with different metal ions, 3 mL of standard solution was taken in a cuvette and then different metal salt solutions (1 × 10 − 3 M) were added accordingly.

Molecular Docking Study
The molecular docking studies of the target protein TRK (PDB ID: 1t46) was carried out with the prepared compound SB-1 and its Cu complex, to elucidate their binding affinity to the ligand binding site of the receptor. Automated docking software AutoDockVina1.2.0 [40,41]. was used for docking calculations that involved rigid receptor docking. A three dimensional model of the structure of the compound and SB-1-Cu was built in Avogadro 3 [42]. Energy minimization was carried out through Force-Filed MMFF94 and optimization was carried out for determining the lowest energy conformation of SB-1 and its Copper complex with most favourable geometry. The crystal structures of c-kit receptor protein-tyrosine kinase in complex with STI-571 (Imatinib or Gleevec) were picked up from the Protein Date Bank (PDB code: 1t46). For the docking calculation, firstly, the protein structure was detached from the co-crystallized ligand STI and hydrogen atoms were added [43]. The area around the predicted ligand binding site on the macromolecule is specified using a grid box. The exhaustiveness parameter that controls the extent of the search was chosen as 32. Nine modes were generated for the ligand. The ligands were flexible in all docking calculations. The validation for molecular docking was done by re-docking the co-crystallized ligand STI in the grid. The predicted docking poses 2D and 3D are then compared with the experimental co-crystallized ligand STI, binding to receptor TRK. OpenBable [44] was used for file format conversions and PyMol [45] for visualization. AutoDockVina output results represented the docking scores as free energy of binding. Protein Ligand Interaction Profiler (PLIP) and Protein Plus was used to identify specific types of interactions in the predicted binding modes such as hydrogen bonds, hydrophobic contacts, and halogen bonds [46,47].