SYNTHESIS, CHARACTERIZATION, LIPOXYGENASE, AND TYROSINASE INHIBITORY ACTIVITIES OF NON-CYTOTOXIC TITANIUM(III) AND (IV) HYDRAZIDE COMPLEXES

. Ti(III) and (IV) hydrazide complexes were synthesized, characterized, and screened for their tyrosinase and lipoxygenase inhibitory and cytotoxic activities. The geometry of Ti(III) hydrazide complexes is tentatively assigned as octahedral. Magnetic moments were found around 1.7 B.M. and electronic spectral transition in the range of 495-518 nm. Evaluation of Ti(IV) and Ti(III) hydrazide complexes for tyrosinase and lipoxygenase inhibitory activities revealed varying inhibition potential. Hydrazide ligands were inactive against tyrosinase, while significant activity was observed against lipoxygenase (LOX). Good to moderate inhibition activity was observed by Ti(IV) and Ti(III) hydrazide complexes against both enzymes. At the same time, promising results were obtained for Ti(IV) hydrazide complexes against tyrosinase enzymes suggesting their broad application as tyrosinase inhibitors. Complex 4d possess negative inhibition, thus behaving as a tyrosinase activator. The docking results showed a good correlation between complex experimental activities and binding energies. Cytotoxic investigation revealed the non-toxicity of complexes against normal cells.


INTRODUCTION
Hydrazides and their transition metal complexes are of astonishing consideration to researchers. They serve as the imperative biological model due to their notable bioactivities, such as antibacterial, herbicidal, antitumor, and antifungal. In hydrazides, C=O (the carbonyl moiety) is suggested to be the responsible component for such activities [1]. Furthermore, like other metal hydrazide complexes, titanium hydrazide complexes have also been reported to possess excellent biological potential like anticancer, antioxidative, photocatalytic, and whitening agent properties [2][3][4]. Therefore, discovering new biologically active titanium complexes is essential to explore their applications as novel drugs.
Cancer has become a severe problem and the second leading cause of death in public healthcare. Chemotherapy using cytotoxic drugs is the most commonly applied treatment to cure this disease. Cytotoxicity is the degree of a compound exhibits a toxic effect on living healthy cells resulting in several cell fates like necrosis, apoptosis, or reduction in cell viability. In a cytotoxic reaction, a non-cytotropic antibody is tagged with foreign antigen on the cell surface, meter (Romania). The degree of magnetization for titanium complexes was measured on Sherwood MSB Mk1 magnetic susceptibility balance using sealed-off MnCl2 solution as calibrant at room temperature. Chloride contents were determined volumetrically using AgNO3 in the presence of indicator potassium chromate solution (1% w/v) [23]. Titanium content in Ti(III) hydrazide complexes was determined volumetrically by the reported method of redox reaction using standard iron(III) ammonium sulfate solution in the presence of potassium thiocyanate solution as an indicator [13].

Synthesis of hydrazide ligands
Hydrazide ligands were synthesized by reported methods by the reaction of hydrated hydrazine with respective ester. First, 50 mmol of hydrated hydrazine was added to an ethanolic solution of ester (10 mmol in 50 mL). Then, the mixture was refluxed for 2-3 h. The products were obtained from crystals separated, washed with hexane, and dried. Ligands were further purified after recrystallization from methanol by slow evaporation.

Synthesis of Ti(III) hydrazide complexes
To synthesize Ti(III) hydrazide complexes, metal and ligand were mixed in 1:2 molar ratios. Then, the methanolic solution of TiCl3 (10 mmol in 10 mL) was slowly added to the synthesized ligands in methanol (20 mmol in 20 mL) with continuous stirring at room temperature. As a result, solid particles were obtained, which were separated and washed with methanol to remove excess reactants. Finally, the precipitates were dried in the air.

Synthesis of Ti(IV) complexes
Our research group has already published the synthesis and characterization of Ti(IV) hydrazide complexes with ligands La-Lf, Lh [24]. The synthesis and structures of Ti(IV) hydrazide complexes are shown in Scheme 1.

Enzyme inhibitory activities
Tyrosinase inhibition assay Tyrosinase inhibition assay was performed in 96 well plate spectrophotometers described by Kim with slight modification [25]. The mixture of 60 units of enzyme, 10 µL of sample in DMSO solution, and 150 µL of buffer (50 mM of pH 6.8) in each well was incubated for 15 min at 30 o C. Then 20 µL of the substrate (L-tyrosine) per well was added and re-incubated at the same condition for 30 min. Dopachrome formed after incubation was determined at 480 nm in a microplate reader. The control comprised 10 µL of DMSO instead of the sample solution. Kojic acid was taken as a positive control for tyrosinase inhibitors. Triplicate sets were used for experiments. Tyrosinase inhibitory percentage was calculated by the formula given below: (Ac -As) x 100 % Inhibition of tyrosinase =  (1) Ac where Ac is the absorbance of the control and As is the absorbance of the sample solution.

Lipoxygenase inhibition assay
LOX inhibition abilities of the samples were determined by the spectrophotometric method described by Tappel et al. with slight modifications [26]. First, the working solution of the LOX enzyme was made and adjusted up to the concentration of the enzyme, displaying the values of 0.05 absorbance/min. Next, 10 µL of test solution (in DMSO) was added to 160 µL (100 mM) of pH 8 sodium phosphate buffer, and then 20 µL of LOX solution was added. The resulting mixture was incubated at 25 o C for 10 min. After incubation, 10 µL substrate solution (linoleic acid, 0.5 mM) was used to initiate the reaction, and the reaction mixture was left for 5 min at room temperature. Then the conversion of linoleic acid to 13-hydroperoxyolinoleic acid was observed, and absorbance was measured at 234 nm using a microplate reader. Finally, the inhibitory concentration of the test compound, at which 50% of the enzyme was inhibited, was determined by varying the concentrations of test compounds using the EZ-Fit Enzyme kinetics program (Perrella Scientific In., Amherst, USA).

Molecular docking
Two target proteins were retrieved from the protein data bank for docking Ti(III) and Ti(IV) hydrazide complexes. The 5-LOX enzyme 3V99 PDB [27] was used, and the tyrosinase enzyme 4OUA PDB [28] was used. Both the proteins were set up for docking by removing water molecules and hetero atoms. In the case of 4UOA protein, chain B was used for docking as it contains copper transition metal at the active site. Similarly, copper metal was retained in chain B of 4UOA. Docking was performed by the online web server PatchDock [29]. Further PLIP [30] online web tool was used to predict protein ligand-complexes interaction.
Initially, Chem draw was used to build the structure of Ti(III) and Ti(IV) complexes. For docking, the Patch Dock server was used with default parameters. For the prediction of binding pattern PLIP online web tool was used to depict protein-ligand complexes interactions. In Patch Dock, the results were evaluated based on atomic contact energy (ACE); a more negative value of ACE shows a greater affinity of the docked compound with the protein. The PLIP web tool was used to evaluate the binding pattern.

Synthesis and physicochemical properties
The hydrazide ligands (La-Lg) were synthesized in good yields (71-85%) and characterized using spectroscopic (IR, EI-MS) and microanalytical (CHN) techniques. Ti(III) hydrazide complexes were synthesized by directly mixing the methanolic solution of TiCl3 with methanolic hydrazide solution in 1:2 ratios at room temperature (Scheme 1, Table 1). Spectral and analytical measurements were applied for the structural elucidation of synthesized complexes (3a-3c, 3e-3g). The experimental section has provided elemental, physical, and analytical data on complexes. Based on the obtained results, the Ti(III) complexes are tentatively assigned to exhibit octahedral geometry displaying the bidentate coordination of ligands to metal. The molar conductivity of Ti(III) complexes in DMSO was 35.26-65.64 Ω 1 cm 2 mol -1 . The values gave the idea of the electrolytic nature of complexes. Molar conductivity values indicate a 1:1 electrolytic ratio of coordination sphere to chloride ion outside the sphere [31]. Complexes were found to be non-hygroscopic and brown in color and showed good solubility in organic solvents like DMSO and DMF. The effective magnetic moments (µeff) of Ti(III) complexes, in the range of 1.71 to 1.78 B.M. suggested the paramagnetic nature of titanium complexes. This value also confirmed the d 1 system and the octahedral geometry of complexes as previously reported for Ti(III) complexes (d 1 system) [32,33]. Despite being oxidation sensitive, Ti(III) did not oxidize to Ti(IV) during complexation, as evident from the obtained spin value.

Spectroscopic analysis
The hydrazide ligands are known to possess two coordination sites, i.e. carbonyl oxygen and amino nitrogen. The binding mode of the hydrazide ligands to Ti(III) in the newly synthesized complexes was evaluated by the exhaustive comparison study of the IR spectra of the ligands with the spectra of Ti(III) complexes. IR spectra of Ti(III) complexes suggest the neutral bidentate coordination of ligands using carbonyl oxygen and hydrazinyl nitrogen. The sharp peak of C=O absorption at 1652±22 cm -1 in ligands shifted to considerable frequency (1668±28 cm -1 ) upon complexation, indicating carbonyl oxygen's involvement in coordination with the metal center. In all complexes, the C=O band shifted to a higher frequency while the C-N band shifted to a lower frequency. The positive frequency shift suggests a decrease in the double bond character of C=O and an increase in the double bond character of C-N, which is supported by the negative shift in C-N frequency [34].
A doublet band around 3030-3322 cm -1 is characteristic of the N-H stretching frequency of the hydrazinyl group. Significant broadening of this band with a lowering of frequency was observed in the spectra upon complexation with Ti(III) center. It supports the coordination of primary nitrogen with the Ti(III) metal center. The bands of benzene skeleton (C=C stretching), NH2 bending, and NH stretching are also identified in the spectra of ligands and complexes.
UV-visible electronic transitions of freshly prepared solution of hydrazide (La-Lc, Le-Lg) and their Ti(III) complexes (3a-3c, 3e-3g) were observed in DMSO. For comparative purposes, electronic transitions of ligands were also listed. The paramagnetic titanium(III) chloride has a d 1 system with 2 T2g ground state and 2 Eg as spin-allowed transitions state. The electronic spectrum of titanium(III) chloride in DMSO showed a prominent band at 525 nm with a shoulder around 575 nm. These transitions are assigned from 2 T2g to 2 B1g and 2 A1g, respectively. All Ti(III) complexes showed one broadband assigned to 2 T2g to 2 Eg with slight shifting in the range of 495-518 nm. The presence of this band confirms the octahedral geometry of complexes [4,35]. Molar absorptivity values (42-73 M -1 cm -1 ) suggested this transition as Laporte forbidden, spin allowed. All ligands exhibit π to π * transition, which comes from the aromatic ring of hydrazide. Upon coordination with Ti(III), these transitions slightly shifted to a lower wavelength which is a sign of lowering the π-orbital energy of the hydrazide ligand.
Based on IR and elemental data, Ti(III) hydrazide complexes are proposed to possess octahedral geometry with neutral hydrazide ligands. The bidentate coordination of ligands was found to have occurred through the oxygen of the carbonyl group and primary amine nitrogen. Ti(IV) hydrazide complexes were also reported to possess octahedral geometries in which the hydrazide ligands are coordinated by the primary amine nitrogen and carbonyl oxygen donor atoms for complexes 4a-4f. In contrast, the imino nitrogen is coordinated in complex 4h [24].

Cytotoxic activities of Ti(III) and Ti(IV) hydrazide complexes
Cytotoxic activity of all Ti(III) and Ti(IV) complexes(100 µM) on normal 3T3 cells have been measured by the appearance of purple color after reduction of MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide) to formazan. Cyclohexamide was used as a standard inhibitor of cytotoxic activity. Most of the complexes found inactive, except 4b, 4c, and 3c showed cytotoxicity much less than standard cyclohexamide. In addition, the precursor metal solutions, TiCl4 and TiCl3, were also found to be non-cytotoxic against normal cells.

Tyrosinase inhibitory activity
Tyrosinase enzyme has a strongly coupled binuclear copper center. It is proposed that inhibitors interact with that binuclear center in the enzyme's active site. Four types of inhibitors can be seen based on their mode of interaction with the enzyme. Inhibitors that compete with substrate and combine directly with free enzymes are competitive inhibitors. Uncompetitive is the one that combines only to enzyme-substrate complex, and the inhibitors bind to both. The enzyme or enzyme-substrate complex with the same and different equilibrium constants are non-competitive and mixed-type inhibitors. Most hydrazides are reported to exhibit mixed-type inhibition [36].
The inhibition potential for all hydrazide ligands and their Ti complexes against tyrosinase enzyme has been investigated by measuring the low absorbance of dopachrome at 480 nm using an L-tyrosine oxidation assay. Kojic acid was taken as the standard tyrosinase inhibitor (IC50 = 25 ± 0.4 µM). The free hydrazide ligands and Ti metal are inactive, while their Ti(IV) hydrazide complexes displayed IC50 values in the range of 4.7 to 151 µM compared to the standard. Specifically, complex 4d displayed enzyme activation. Carbonyl hydrazide moiety could form a complex with the active site copper center of tyrosinase enzyme, thus taking an important part in inhibition [17,37,38]. Four out of six Ti(IV) complexes, including 4a, 4e, 4f, and 4h, showed the most potent inhibitors of tyrosinase enzyme in comparison to the standard Kojic acid. An unsubstituted 4a (IC50 = 4.7 ± 0.3 µM) complex with only a benzene ring of hydrazide ligand was the most potent inhibitor and five-fold more active than the standard. Incorporation of halides e.g. Cl and I at para positions of benzene ring in cases of 4b (IC50 = 140.0 ± 0.2 µM) and 4c (IC50 = 151.0 ± 0.4 µM) lead to steep decline in the inhibitory potential ( Figure 1).
However, Ti complex 4d having ortho-fluoro substitution at the benzene ring exhibits a negative inhibition value showing the enzyme activating property. For effectors binding, an effector site is also present in the tyrosinase enzyme in addition to the active site [39]. As reported for sodium dodecyl sulfate (SDS), limited conformational change in latent enzyme after binding to SDS is related to the activation process [40][41][42]. Fatty acid produces conformational change and hence showed activation of tyrosinase enzyme. Since fluoro-carbon compounds are hydrophobic [43], in our research, it can be proposed that the hydrophobic fluoro-carbon can bind to the effect or site of the enzyme with a strong hydrophobic attraction leading the conformational change in the active site of the enzyme. This conformational change makes the active site more effective than the substrate, resulting in activation (Figure 1).
Pyridyl ring bearing Ti(IV) complexes 4e (IC50 = 5.8 ± 0.5 µM) and 4f (IC50 = 7.9 ± 0.4 µM) demonstrated potent inhibitory potential against tyrosinase enzyme. Studies revealed that replacing the benzene ring with an electron-rich aromatic moiety favors the binding of the inhibitor with the active site of the enzyme resulting in better inhibition potential [40]. Recent studies also described the role of pyridyl nitrogen of inhibitors in the complex formation with the copper center in the enzyme's active site [44,45]. Minor difference in results indicates the positional effect of pyridyl N of a benzene ring on inhibition. Complex 4h (IC50 = 5.4 ± 0.3 µM) possesses the second most potent activity among all the complexes (Figure 1). The hydrazide ligand contains an imino N in addition to the hydrazinyl N, thus forming the electron-rich complex which favors the inhibition potential. The Ti complex (4h) is most symmetric in structure as the Ti center is coordinated to three same bidentate ligands giving perfect Oh geometry. The symmetric structure may also be the reason for the high potency since the significant effect of molecular symmetry on enzyme inhibition is already reported [46]. TiCl3 itself is found to be inactive, while the complexes are found to be active against tyrosinase enzyme. Ti(III) hydrazide complexes exhibited varying degrees of tyrosinase inhibition displaying the same activity trend as discussed for Ti(IV) hydrazide complexes. The simplest unsubstituted complex 3a (IC50 = 5.6 ± 0.3 µM) is found to be the most potent amongst all complexes, while the substitution of halo groups, i.e. chloro in 3b (IC50 = 160.0 ± 0.4 µM) and iodo in 3c (IC50 = 178.0 ± 0.2 µM) at the para position of the benzene ring, decreased the activity of complexes and exhibited weak inhibition. Pyridyl ring complexes, 3e (IC50 = 7.8 ± 0.5 µM) and 3f (IC50 = 18.0 ± 0.3 µM) displayed increased inhibition potential exhibiting IC50 values (7.8 and 18 µM, respectively) better than the standard Kojic acid. The electron-donating groups are reported to exhibit more potency than halogen substitution on the benzene ring [47]. Complex 3g (IC50 = 63.0 ± 0.4 µM), with residual methylene (-CH2) in between the carbonyl group and benzene ring of ligand, displayed good inhibition, suggesting the effective involvement of the CH2 group in enhancing the inhibitory potential against tyrosinase enzyme (Figure 2).

Docking analysis
The docking analysis confirms that hydrazide compounds are inactive against the tyrosinase enzyme and do not bind at the active site of the tyrosinase enzyme. Figure 3 shows that all hydrazide ligands have similar binding patterns with His4 and His5 amino acid residues but not lying at active sites and have very low ACE scores compared to complexes of hydrazide. The results validate that hydrazide compounds are not binding at the binding pocket and are inactive against the tyrosinase enzyme's experimental activity. However, the estimation of the binding mechanism of Ti(III) and Ti(IV) complexes results in a variable degree of docking binding energies. In the case of Ti(III) complexes (Figure 4), the most potent complex is 3a and 3e for tyrosinase enzyme, where docking results also show the highest binding affinity with this complex at the active site owing -235.56 cal/mol and -224.5 cal/mol, respectively. They form a hydrogen bond with GLN294 at a distance of 1.98 Å and 3.14 Å. In addition, complex 3e also forms a hydrogen bond with SER291 and GLY299 at a distance of 3.06 Å and 2.78 Å, respectively. In the case of complex 3f, binding energies follow a similar pattern with experimental activities and form hydrogen bonds, including SER291, GLN294, GLY299, THR343, ASP344, THR345, ALA346, and SER351 at a distance of 3.43, 2.24, 3.14, 3.16, 2.56, 2.15, 3.20, and 2.57 Å, respectively. Complex 3g has binding energy -183.79 cal/mol mediating hydrogen bond with GLN 294 at a distance of 3.48 Å, and it also forms water bridge interaction with ALA346 at a distance of 3.76 Å. Complexes 3b and 3c have low experimental activity and mediate weak hydrogen bonds with SER439 and GLN294, respectively. In the case of Ti(IV) complexes ( Figure  4), compounds 4a, 4h, 4e, and 4f show high experimental activity and good binding affinity with active site amino acid residues, including LYS66, THR70, GLN294, THR343, ASP345, ARG376. In addition, complex 4h also mediates polar contact with surrounding water molecules by making a water bridge with ALA346 at a distance of 3.06 Å. It also has π-stacking interactions with PHE355. Complexes 4b and 4c have low experimental activity, so compound 4b does not interact with surrounding amino acids, whereas compound 4c only forms a bond with THR70 at a distance of 2.18 Å. Complex 4d acts as an experimental activity activator and forms bonds with SER 291 at a distance of 2.79 Å.

Lipoxygenase inhibitory activities
The inhibition potential of the hydrazides, their Ti(III) and (IV) complexes, and precursor metal solutions (TiCl3 and TiCl4), have been screened for lipoxygenase inhibition activity. All the hydrazides and their respective titanium complexes showed varying degrees of LOX inhibition, while the metal solutions (TiCl3 and TiCl4) possess no activity. Baicalein was the standard lipoxygenase inhibitor with an IC50 value of 27 ± 0.4 µM. The non-coordinated hydrazides exhibited superior LOX inhibition in the range of 28-55 µM compared to the metal complexes and standard inhibitor. It may be due to the ability of the ligand to coordinate with the active site of the enzyme, Fe +3 , and reduce it to the inactive Fe +2 [48,49]. The hydrazide Lc (IC50 = 28 ± 0.3 µM) with unsubstituted benzyl ring is the most potent inhibitor among all the hydrazides compared to the standard. The substitution of halide groups decreases the inhibition activity of ligands depending upon the nature and position of the substituent. parachloro and para-iodo bearing ligands Lb (IC50 = 45 ± 0.4 µM) and Lc (IC50 = 40 ± 0.3 µM) displayed comparatively better activity in comparison to ortho-fluoro substituted Ld (IC50 = 55 ± 0.2 µM). Replacement of benzene ring with 3-pyridyl Le (IC50 = 28 ± 0.3 µM) and 4-pyridyl Lf (IC50 = 29 ± 0.5 µM) ligands display no change in the LOX inhibition activity having results similar to benzyl ring hydrazide. Imino moiety in between the carbonyl group and benzene ring of hydrazide ligand Lh (IC50 = 40 ± 0.5 µM) positively affects inhibition activity. The presence of the CH2 group in between carbonyl and benzene ring in hydrazide ligand Lg (IC50 = 47 ± 0.6 µM) results in a lowering of LOX inhibition activity.
Ti(IV) hydrazide complexes exhibited moderate LOX inhibitory activity in the range of IC50 = 58 to 99 µM. The presence of titanium metal may hinder the coordination of hydrazide with iron situated at the active site of lipoxygenase, thus lowering the activity of complexes compared to the free hydrazide ligands. The pyridyl ring complexes 4e (IC50 = 58 ± 0.5 µM) and 4f (IC50 = 60 ± 0.6 µM) possess the noteworthy inhibition potential amongst all Ti(IV) complexes. Complex 4h (IC50 = 63 ± 0.3 µM) with imino moiety in between the carbonyl group and benzene ring also displayed imperious LOX inhibiting activity. The results suggest nitrogen moiety's operative role in enhancing the LOX inhibition potential of Ti(IV) hydrazide complexes 4e, 4f, and 4h. It is worth mentioning that the same complexes possess efficient antioxidative properties against DPPH. Since the compounds possessing DPPH scavenging potential exhibit remarkable antiinflammatory and antiaging properties [50]. Unsubstituted 4a (IC50 = 88 ± 0.3 µM), parachloro 4b (IC50 = 99 ± 0.2 µM), para-iodo 4c (IC50 = 85 ± 0.2 µM), and ortho-fluoro 4d (IC50 = 87 ± 0.4 µM) hydrazide complexes demonstrated moderate and closed inhibitory activities ( Figure  5). LOX inhibition activity of Ti(III) hydrazide complexes was found in the range of 57 -89 µM, lower than hydrazide ligands. Complex 3g, with the CH2 group in between C=O and benzene ring, displayed no inhibition activity against the LOX enzyme, while the respective free ligand exhibited good activity. The low activity in the case of complexes supports our hypothesis that the coordination of hydrazide and active site iron is hindered in the presence of Ti(III) metal. The complex with unsubstituted benzene ring 3a (IC50 = 79 ± 0.4 µM) showed moderate inhibitory potential than the standard baicalein (IC50 = 27 ± 0.4 µM). The increase in activity was observed after the substitution of the iodo group at para position 3c (IC50 = 57 ± 0.6 µM) while replacing iodo with the chloro group in the case of complex 3b (IC50 = 89 ± 0.5 µM) resulted in decreased inhibitory potential. The pyridyl bearing complexes 3e (IC50 = 74 ± 0.2 µM) and 3f (IC50 = 69 ± 0.4 µM) also displayed moderate activities. No marked effect was observed with respect to the position of pyridyl nitrogen ( Figure 6).

Docking studies
Docking binding patterns of hydrazides are already reported by our research group [22], while their Ti(III) and Ti(IV) complexes have been estimated for docking binding energies. In case of Ti(III) complexes (Figure 7), the most potent compound, 3c, shows binding energy of -171.41 cal/mol. It forms two hydrogen bonds with GLU108 and GLU134 at a distance of 2.28 and 3.32 Å. In addition, it also forms a halogen bond with TYR142 at a distance of 2.42 Å and mediates πcation interactions with ARG101. In the case of compounds, 3a and 3e binding energies follow a similar pattern with experimental activities, and both 3a and 3e form bonds with ASN216, SER608, and ARG666. In the case of compound 3b, experimental activity is low among all compounds, and it also has low binding energy, i.e., -156.18 cal/mol as compared to other compounds, and only forms bonds with ARG 101 and THR137. For compound 3g, lipoxygenase activity is nil, so docking energy is very low, i.e. -83.31 cal/mol. For compounds 4e and 4h, lipoxygenase activity is high, i.e. 58 µM and 63 µM, respectively (Figure 7), so binding energies are also higher, i.e. -197.99 and -189.13 cal/mol. In the case of compounds 4a, 4c and 4d, experimental activity is 85-89 µM. Compound 4a only shows hydrophobic interactions where compound 4c forms a hydrogen bond with ASN554 at a distance of 2.46 Å. For compound 4d, it forms a hydrogen bond with TYR558 and ASP559 at a distance of 3.13 and 3.10 Å. Compound 4b has low experimental activity and low binding energy and only forms a bond with VAL 110 at 3.28 Å. The current findings suggested that hydrazide complexes of Ti(III) and Ti(IV) bind effectively with the target enzyme, and this study further can be used for therapeutic targets.

CONCLUSION
Hydrazide ligands and their Ti(III) and Ti(IV) complexes were synthesized and characterized through chemical and physical measurements. Enzyme inhibition investigation of free hydrazide ligands and their respective Ti(III) and Ti(IV) complexes displayed a varying degree of tyrosinase and lipoxygenase inhibitory potential. The free hydrazides showed potent LOX inhibitory activity compared to the respective titanium complexes. Most of the titanium complexes exhibited good inhibitory potential against tyrosinase enzyme and hence can be used for hyperpigmentation problems, while compound 4d is an enzyme activator. All Ti-hydrazide complexes are found noncytotoxic against normal 3T3 cells. The foregoing activity trail of Ti-complexes suggested that the substituent modifications in the structure of hydrazide can help to explore the novel leads for antiinflammatory activity and treat melanin disorders. The non-toxic behavior of Ti(III) and Ti(IV) complexes to normal 3T3 cells also encourages the detailed study of novel titanium-based anticancer drugs against human cancer cells.