Copper(II) chloride (0.89 g, 0.005 M) was dissolved in aqueous ethanol. A solution of ligand L (5.92 g, 0.01 M) was obtained by dissolving it in warm ethanol, 30 ml hot buffer (ammonium acetate) solution at pH 7 was then added to this with stirring. The resultant solution was added drop-wise with vigorous stirring to the Copper(II) chloride solution. The reaction mixture was then refluxed for 4 h. The resulting mixture was then cooled. A pale-yellow precipitate was obtained. This was recrystallized using methanol/water mixture (10 mL).

Yield: 2.46 g, 72.28%; m.pt: 162˚C - 163˚C (d); metal composition (%): calc.: 9.37; found: 10.24; µ_eff: 1.98 BM. The product obtained was soluble in water and insoluble in ethanol, methanol.

Similar procedure was adopted for the syntheses of the following complexes.

Manganese(II) chloride tetrahydrate (1.01 g, 0.005 M) and ligand L (5.92 g, 0.01 M) afforded a pale orange precipitate. Yield: 2.23 g, 65.28%; m.pt: 180˚C - 182˚C; metal composition (%): calc.: 9.51; found: 9.87; µ_eff: 5.82 BM. The product obtained was soluble in water and insoluble in ethanol, methanol.

Zinc(II) chloride (0.18 g, 0.005 M) and ligand L (5.92 g, 0.01 M) afforded an orange precipitate. Yield: 2.21 g, 69.45%; m.pt: 250˚C - 251˚C (d); metal composition (%): calc.: 8.62; found: 9.28. The product obtained was soluble in water and insoluble in ethanol, methanol.

Nickel(II) chloride hexahydrate (1.21 g, 0.005 M) and ligand L (5.92 g, 0.01 M) afforded a light blue precipitate. Yield: 2.35 g, 69.23%; m.pt: 248˚C - 249˚C (d); metal composition (%): calc.: 8.71; found: 8.56; µ_eff: 2.98 BM. The product obtained was soluble in water and insoluble in ethanol, methanol.

Cobalt(II) chloride hexahydrate (1.19 g, 0.005 M) and ligand L (5.92 g, 0.01 M) afforded a light brown precipitate. Yield: 2.64 g, 71.85%; m.pt: 201˚C - 202˚C (d); metal composition (%): calc.: 9.19; found: 9.54; µ_eff: 2.26 BM. The product obtained was soluble in water and insoluble in ethanol, methanol.

The Xanthine Oxidase (XO) inhibitory activity of ligand L and compounds 1-5 was assayed spectrophotometrically under aerobic conditions as described by Nguyen et al. 2006, with some modifications [40]. The assay was carried out at five different concentrations ranging from 0.03125, 0.0625, 0.125, 0.25, 0.5 to 1 mg/L for the samples. These were directly dissolved in phosphate buffer-MeOH (1%) and screened for xanthine oxidase inhibitory activity at final concentration of 50 µg/mL. The mixture assay consisted of 150 µL phosphate buffer (1/15 M, pH 7.5), 50 µl sample solution and 50 µl enzyme solution (0.28 units/mL in phosphate buffer). The reaction was initiated by adding 250 µL of substrate solution (0.15 mM in water). Enzymatic kinetic was recorded at 295 nm for 02 min. The negative control was prepared and consisted of 1% methanol solution without the samples. Allopurinol a well-known inhibitor of xanthine oxidase was used as a positive control at a final concentration of 50 µg/mL. All experiments were performed in triplicate. Xanthine oxidase inhibitory activity was expressed as the percentage inhibition of xanthine oxidase, calculated as (%) inhibition following Equation (1) and the IC₅₀ values were determined. The IC₅₀ values were calculated from the mean values of data from four determinations.

( % ) inhibition = ( 1 − B / A ) × 1 00 (6)

where:

A is the change in absorbance of the assay without the sample (Δabs. with enzyme-Δabs. without enzyme);

B is the change in absorbance of the assay with the sample (Δabs. with enzyme-Δabs. without enzyme);

NB: 1 unit of XO is defined as the amount of enzyme required to produce 1 µmol of uric acid/min at 25˚C.

Lipoxygenase inhibitory activity of ligand L and compounds 1-5 with linoneic acid as substrate was measured with a UV/visible spectrophotometer as described by Gunathilake et al., 2018, with some modifications [15]. The assay was carried out at five different concentrations ranging from 0.03125, 0.0625, 0.125, 0.25, 0.5 to 1 mg/L for the samples. The samples were screened for lipoxygenase inhibitory activity at final concentration of 50 µg/mL. The assay mixture consisted of 150 µl phosphate borate (1/15 M, pH 7.5), 50 µL of each sample solution and 50 µL lipoxygenase solution (0.28 U/ml in phosphate borate). The reaction was initiated by adding 250 µL of linoneic acid solution (0.15 mM in water). Enzymatic kinetic was recorded at 234 nm for 02 min. The negative control contained 1% methanol solution, without sample solution, to the above mixture. All experiments were performed in triplicate. Lipoxygenase inhibitory activity was expressed as the percentage inhibition of lipoxygenase, calculated as (%) inhibition following Equation (6). Quercetin was used as the positive reference drug. The IC₅₀ values were calculated from the mean values of data from four determinations.

1) Preparation of Red Blood Cells (RBC)

The bovine red blood cell was prepared according to the method of Mohamed et al., 2014 [16]. Fresh bovine blood was mixed with an anti-coagulant (3.8% tri-sodium citrate) in a clean bottle in ratio 2:1 (blood: anti-coagulant) and mixed gently by inversion. The blood sample was centrifuged at 3000 rpm on a bench centrifuge, Model 90-2, for 10 min. at room temperature. The supernatant layer (plasma and leucocytes) was carefully removed while the packed red blood cell was washed again in fresh normal saline (0.85% w/v NaCl). The process of washing and centrifugation was repeated several times until the supernatant layer was clear (colourness). Bovine erythrocytes (2% v/v) were then prepared by diluting 2 mL of packed cell with normal saline to 100 mL. This was kept undisturbed at 4˚C in the refrigerator.

2) RBC Membrane Stabilization Assay

The membrane stabilizing activity for ligand L and compounds 1-5, was carried out according to the modified procedure as described by Oyedapo et al., 2010 [41]. The assay mixture consisted of hyposaline (1.0 mL), 0.15 M phosphate buffer, pH 7.4 (0.5 mL) of varying volumes of solutions of the samples (0 - 1000 µg), 0.5 mL of 2% (v/v) erythrocyte to give a final volume of 3.0 mL. The reaction mixture was incubated at 56˚C for 30 min. The tubes with the mixture were then cooled and centrifuged at 3000 rpm for 10 min. The supernatants obtained were collected and the absorbance was monitored at 560 nm against a reagent blank.

The percentage stability was estimated as follows:

% membrane stability = { 100 − ( Abs of test drug − Abs of drug control ) Abs of blood control ( blank ) − Abs of drug control × 100 } (7)

The blood control used was diclofenac, this represents 100% lysis.

Protein (albumin) denaturation assay was done according to the method described by Gambhire et al., 2009 [42], with some modifications as described in Gunathilake et al., 2018 [15]. The synthesized compounds and positive standard (diclofenac) were prepared at a concentration of 0.1% each (1.0 mg/ml). The reaction mixture (5 mL) consisted of 0.2 mL of 1% bovine albumin, 4.78 mL of phosphate buffered saline (PBS, pH 6.4), and 0.02 mL sample of the synthesized compounds, and the mixture was mixed, and was incubated in a water bath (37˚C) for 15 min, and then the reaction mixture was heated at 70˚C for 5 min. After cooling, the turbidity was measured at 660 nm using a UV/VIS spectrometer (Optima, SP-3000, Tokyo, Japan). Phosphate buffer solution was used as the control. The percentage inhibition of protein denaturation was calculated by using the following formula:

% inhibition of denaturation = 1 00 × ( 1 − A 2 / A 1 ) (8)

where A₁ = absorption of the control sample, and A₂ = absorption of the test sample.

The FTIR spectrum of ligand L (Figure 4) exhibited a weak broad band at 3529 cm⁻¹ ascribable to the stretching absorption frequency of the hydroxyl substituent [43] [44] [45]. This was further supported by bands observed at 1379 and 1148 cm⁻¹ which were assigned to δ(O-H) and ν(C-OH) absorption frequencies respectively [43] [44] [45]. Weak extended sharp bands in the high energy region of the spectrum suggest hydrogen bonding. This may be attributed to intermolecular interaction between phenolic substituents of molecules of ligand L. The spectrum was devoid of absorption band attributable to the amino (-NH₂) substituent present in 2-amino-4,5-dimethylthiazole, one of the reactants. This therefore suggest the diazotization of the heterocyclic amine and plausible coupling of this with 5-methyl-2-(propan-2-yl)phenol. This was however confirmed by the appearance of the N=N absorption band observed at 1446 cm⁻¹ (Table 1) [43] [44] [45]. The appearance of this absorption band at the observed frequency, lower than that expected, suggests conjugation with the heterocyclic imine at one end and the aromatic phenol at the other end [43] [44] [45]. It should be noted that this frequency was absent in the spectrum of all the reagents. Additional evidence for the formation of the thiazolylazo dye ligand was obtained as a result of the band observed at 1543 cm⁻¹ attributable to ν(NN) + ν(NC) [43]. Corroborating this further was the ν(C=N) at 1576 cm⁻¹ [43] [44] [45].

The electronic spectrum in the ultraviolet region exhibited three intense bands at 290, 324 and 477 nm (Table 2) ascribable to n→σ*, π→π* and n→π* of the major chromophores of the ligand; the phenolic -OH, C=N and N=N substituents [44] [45]. The first band is assignable to transition in the phenolic substituent [44]. The second transition is suggested to originate from the imine substituent which is in conjugation thiazole ring as well as the azo moiety [45]. The third band is attributable to transition due to the N=N substituent in addition to intermolecular charge transfer taken from the phenolic ring through the azo group [45].

The Fourier transformed infrared spectrum of the complex exhibited two broad bands at 3529 and 3347 cm⁻¹ attributable to ν(O-H) (Table 1) [43] [44] [45]. The former frequency occurred at the same frequency as the free base. This therefore suggests that coordination did not occur via this functional group within the ligand [43]. The latter frequency occurred lower than that observed for the free base. This is suggestive of coordination of solvent from the reaction medium. This was corroborated with the frequency bands observed at 1185 and 1043 cm⁻¹ ascribed to δ(O-H) and ν(M-OH) for the solvent. According to Pavia et al. 2001, a distinguishing feature between a phenolic and other hydroxyl substituent is the frequency at which the δ(O-H) and ν(C-OH) are observed [44]. For phenolic -OH the δ(O-H)Ar and ν(C-OH) are observed at higher energy as a result of the oxygen atom in conjugation with the double bond from the ring. In this case both δ(O-H) and ν(C-OH) were observed and the one observed at higher frequency at 1367 cm⁻¹ was ascribed to the δ(O-H), with the lower frequency at 1110 cm⁻¹ due to ν(C-OH) of the phenolic moiety [43] [44] [45]. Bands observed at 674 and 607 cm⁻¹ were attributed to ν(M-O) [46] [47] [48]. This further supported the coordination of oxygen atoms of the solvent molecule to the metal ion [43]. Evidence for the coordination of the imine moiety was provided by the band observed at 1558 cm⁻¹ ν(C=N). A shift of 80 cm⁻¹ was observed for the ν(N=N) in comparison with the free ligand as a consequence this suggested coordination of a nitrogen atom of the azo functional group [43]. Additional evidence for the coordination of nitrogen atoms to the metal ion was provided by the frequency band observed at 540 cm⁻¹ attributable to Cu-nitrogen stretching frequency [49].

The electronic spectrum of compound 1 elicited six bands. In the ultra violet region, the spectrum displayed three intense bands at 285, 373 and 487 nm. These were ascribed to n→σ*, π→π* and n→π* intraligand transitions of the major chromophores of the ligand [43] [44]. This is indicative of a bathochromic shift for the least wavelength and hysochromic shifts for the other two wavelengths. As a result, it suggests possible coordination of ligand L to the metal ion [43]. In the visible region a shoulder was observed at 525 nm and assigned to ²B_1g→²A_1g transition [50] [51]. In addition to this, two bands were also observed at 683 and 884 nm and attributed to ²B_1g→²B_2g and ²B_1g→²E_g d-d transitions [50] [51]. It should however be noted that the electronic spectrum of ligand L did not exhibit absorption bands in the visible region. This appearance of new absorption bands, in the visible region of the spectrum, which were absent in the ligand’s, indicated coordination of the metal ion with ligand L [43]. The transitions are consistent with a Jahn Teller distorted octahedral geometry around Cu(II) ion. The magnetic moment obtained at 1.98 BM, indicative of a tetragonally distorted octahedral geometry for a 3d⁹ configuration for a Cu(II) ion, further affirmed this geometry [52].

The FTIR spectrum for compound 2 (Figure 5) displayed two bands at the high energy region. A weak band was observed at 3794 cm⁻¹, the second was a broad band with the shape of a trough observed at 3406 cm⁻¹. The first band may be ascribed to the phenolic ν(O-H) with intermolecular hydrogen bonding [43] [44] [45]. This was corroborated by the δ(O-H)Ar observed at 1379 cm⁻¹ and ν(C-OH) at 1148 cm⁻¹. The lower energy band may be ascribed to ν(O-H) from water the reaction medium as a result of its shape [43] [44] [45]. This was supported by δ(O-H) and ν(C-OH) frequency bands observed at 1181 and 1051 cm⁻¹. It was further corroborated by the appearance of two bands in the far infrared at 674 and 629 cm⁻¹. Attributed to (Mn-O) vibrational frequency [49]. The ν(N=N) was observed at 1435 cm⁻¹ indicative of a bathchromic shift in comparison with that obtained in the free ligand and therefore suggested coordination of one of the nitrogen atoms of the azo moiety in the ligand to the metal ion [43]. This was supported by the band observed at 570 cm⁻¹ ascribed to the ν(Mn-N). Additionally, two bands with medium intensities were observed at 1636 and 1532 cm⁻¹ and were assigned to ν(C=N) and ν(NC) + ν(NN). The former stretching frequency indicated a hysochromic shift of 60 cm⁻¹ in comparison with the ligand. Therefore, it points to the fact that the azomethine nitrogen atom present within the thiazole ring participated in coordination of the metal ion with the ligand [43].

The electronic spectrum of this compound exhibited two intense bands at the ultraviolet region at 230 and 320 nm (Table 2). Assigned to n→σ* and π→π* intraligand transitions [44] [45]. Three bands with low intensities were observed at the low energy region at 521, 617 and 864 nm. These were attributed to ⁶A_1g→⁴E_1g(G), ⁶A_1g→⁴T_1g(G) and ⁶A_1g→⁴T_1g(G) d-d transitions of the central metal ion [50] [51]. The transitions are in agreement with an octahedral geometry for a d⁵ configuration for Mn(II) [49] [50] [51].

According to Greenwood and Earnshaw, the explanation of these features stems from the fact that any electronic d-d transition from a high spin d⁵ configuration must of necessity involve the pairing of some electron spins [51]. It follows therefore that such transitions are spin and orbital forbidden and that the absorptions should be of extremely low intensity [50] [51]. That they occur at all is due to weak spin-orbit coupling. The magnetic moment of 5.82 BM, indicative of high spin 3d⁵ configuration for Mn(II), this therefore further validates the octahedral geometry for this compound [49] [50] [51].

The infrared spectrum of this compound (Figure 6) showed a broad band at 3391 cm⁻¹ corresponding to ν(O-H) of the phenol substituent [43] [44] [45]. This was supported by bands observed at 1379 and 1054 cm⁻¹ which were assigned to δ(O-H)Ar and ν(C-OH) [43] [44] [45]. In this case, a 90 cm⁻¹ difference exists between the observed ν(C=N) and that obtained in the ligand. This therefore suggested coordination of the lone pair of electrons bearing nitrogen atom of the imine substituent to the metal ion [43]. This was supported by the absorption band at 1241 cm⁻¹ attributable to δ(C-N). Evidence for the coordination of one of the nitrogen atoms of the azo moiety was provided by the frequency band observed at 1438 cm⁻¹ ascribed to the ν(N=N) [43]. Further support for the coordination of the nitrogen atoms of the ligand was given by the band at 521 cm⁻¹ attributable to the ν(M-N) [43].

Two intense bands were observed in the electronic spectrum of this complex at 320 and 340 nm (Table 2). Consistent with π→π* and n→π* intraligand transitions [44] [45]. No distinctive band was observed in the visible region of this spectrum [50] [51]. This is not unexpected as the central metal ion has a completely filled 3d orbital i.e. d¹⁰ configuration. The magnetic susceptibility measurement for compound 3 showed a diamagnetic behavior. This is in agreement with previous established reports [50] [51].

The FTIR of this compound (Figure 7) revealed weak extended bands at the high energy region of the spectrum with a weak peak at 3794 cm⁻¹ ascribed to the phenolic ν(O-H), with intermolecular hydrogen bond [43] [44] [45]. This was supported by the bands at 1323 and 1114 cm⁻¹ which correspond to δ(O-H)Ar and ν(C-OH) [43] [44] [45]. The (C=N) frequency band was obtained at 1636 cm⁻¹, the blue shift in comparison with the ligand serve to suggest coordination

of the nitrogen atom to the metal ion [43]. This was supported by the band observed at 1569 cm⁻¹ ascribable to ν(NC) + ν(NN) [43]. Additional support, was obtained in the δ(C-N) with a shift of 56 cm⁻¹, serving as evidence for coordination of the nitrogen atom of the imine substituent [43]. In addition to this, the ν(N=N) was observed at lower energy in comparison to the ligand at 1435 cm⁻¹. This suggests shortening of the bond and as a consequence it indicated coordination of the ligand via one of the nitrogen atoms of this substituent to the metal ion [43]. This was further supported by the ν(NN) + ν(NC) at 1490 cm⁻¹. Further evidence was provided by the ν(Ni-N) frequency band at 570 and 529 cm⁻¹ [49]. The spectrum also displayed two medium and broad absorption bands at 3175 and 3060 cm⁻¹ with the latter shaped as a trough [43] [44] [45]. These may be attributed to absorption frequencies due to a hydroxyl substituent as a result of its shape with overlap with the absorption frequency for sp³ hybridized carbon-hydrogen bond [43] [44] [45]. According to Pavia et al., 2001, the frequency at which this was observed suggests the presence of hydrogen bonding [44]. This was further supported by the appearance of bands at 1203, 1185 and 1051 cm⁻¹ attributable to δ(O-H), ν(C-O) and δ(M-O-H). They therefore suggest coordination of the metal ion with the reaction solvent. Buttressing this were the bands observed at 670 and 629 cm⁻¹ which are due to nickel-oxygen stretching frequencies [43] [49].

Three intense bands were observed in the ultraviolet region of the electronic spectrum for this compound. They were observed at 209, 309 and 372 nm (Table 2). Attributed to n→σ*, π→π* and n→π* intraligand transitions [44] [45]. Two broad bands were observed at 460 and 769 nm in the visible region of the spectrum. These are ascribable to ³A_2g(F)→³T_1g(P) and ³A_2g(F)→³T_2g(F), d-d transitions [50] [51]. Indicative of an octahedral geometry [50] [51]. Corroborating this is its magnetic moment of 2.98 BM [49] [50] [51].

Extended sharp bands with a weak band was observed in the high energy region of the Fourier transformed infrared spectrum of the complex. The band at 3585 cm⁻¹ is attributable to the vibrational frequency due to hydroxyl substituent in the ligand [43] (Figure 8). In support of this was the band at 1379 cm⁻¹ ascribed to δ(O-H)Ar. The extended bands are suggestive of hydrogen bond relatable to the hydroxyl moiety [44]. In this complex the ν(N=N) gave a bathochromic shift of about 23 cm⁻¹ in relation with the ligand. This suggest the elongation of the bond, thereby it may serve as evidence of coordination of the ligand to the metal ion [43]. Corroborating this further was similar shift observed in the ν(C=N) to 1572 cm⁻¹. The metal-nitrogen bond observed at 555 cm⁻¹ also stands to confirm the coordination of a nitrogen atom with the cobalt(II) ion. Additional evidence was provided by ν(NC) + ν(NN) absorption frequency at 1543 cm⁻¹. Bands observed at 1107 and 1047 cm⁻¹ were ascribed to δ(O-H) and ν(M-OH) suggest coordination of water molecules. The ν(Co-M) observed at 682 cm⁻¹ corroborates this further.

In this present case the electronic spectra elicited three intense bands at 265, 323 and 422 nm. These may be ascribed to n→σ*, π→π* and n→π* intraligand transitions of the major chromophores of the ligand [44] [45]. Additionally, a broad band was observed near infrared region at 815 nm which may be assigned as ⁴T_1g(F)→⁴T_2g(F) [50] [51]. A magnetic moment of 2.26 BM was obtained for compound 5. This may be interpreted according to previous reports to be attributable to four-coordinate, square planar geometry for a cobalt(II) complex [50] [51] [53] [54].

The diazonium salt obtained with 2-amino-4,5-dimethylthiazole reacted with 5-methyl-2-(propan-2-yl)phenol to give the thiazolylazo dye ligand L (Scheme 1). A polyfunctional ligand, bearing substitutes with lone pair of electrons viz: -OH, C=N and N=N substituents, with potential donor atoms. From the results

obtained, in the case of compounds 1, 2 and 4 two bands were observed at the high energy region of their spectra and were assigned to the ν(O-H). Initially, one may tend to ascribe both to the asymmetric and symmetric stretching modes for the hydroxyl substituent. However, further analysis of the spectra suggested the presence of water molecule within the coordination sphere of the metal ions. This was alluded to by the presence of absorption frequencies attributable to the δ(O-H) and ν(M-OH). The results obtained from the FTIR spectra, was further corroborated by data obtained from their electronic spectra, magnetic susceptibility measurements and metal analyses. Therefore, based on these an octahedral geometry is proposed for these compounds. This is depicted in (Figure 9). In similar vein, the spectrum of compound 5 exhibited only one band for the ν(O-H), it was attributed to the stretching vibrational frequency of the phenolic substituent. Although, in addition to this, frequency bands ascribable to δ(O-H), δ(O-H)Ar and ν(M-OH) were observed. According to Nakamoto et al., 2009, water in inorganic salts may be classified as either lattice or coordination water [43]. There is, however, no definite borderline between the two. The former term denotes water molecules trapped in the crystal lattice, either by weak hydrogen bonds to the anion or by weak coordinate bonds to the metal, or by both. In this case we propose that the water molecule, as suggested from the FTIR spectrum, is lattice water [43]. This is in agreement with that obtained from the electronic spectrum, magnetic moment and percentage metal analysis. Therefore, compound 5 is proposed to assume a square planar geometry (Figure 10). For compound 3, the Zn(II) complex, the FTIR spectrum exhibited a band for the ν(O-H), δ(O-H)Ar and ν(C-OH). Contrary to the other compounds, in this case it is interesting to note that similar absorption bands due to coordination of water was absent in this spectrum. The electronic spectrum and magnetic susceptibility measurement data indicated that the complex is diamagnetic. This is not unexpected, as a result of the fully filled penultimate, 3d shell. This in collaboration

with the FTIR and percentage metal analysis suggest a four-coordinate geometry for this compound. Indicating either a tetrahedral or square planar geometry for this complex. Theoretically a tetrahedral geometry is more readily assumed by this metal ion, to afford sp³ hybridization [50]. However, a square planar geometry is proposed (Figure 10). This is because of the nature of the ligand and more importantly the limited flexibility along the substituents bearing the potential donor atoms.

Additionally, the results obtained suggest that the ligand chelated with the metal ion as a bidentate ligand. It coordinated to the metal ion through the nitrogen atom of the imine substituent and via one of the nitrogen atoms of the azo moiety. Theoretically, coupling of the phenol to the diazonium ion ought to take place at either the ortho or para position, with the ortho position taking precedence if available. The result obtained suggested that this was not the case for ligand L (Figure 3). It is proposed that the diazo-coupling took place at the para position to the phenol substituent in the benzene nucleus of 5-methyl-2-(propan-2-yl)phenol as a result of steric effect.

According to Borges et al., from rheumatological diseases point of view, Xanthine Oxidase (XO) is an enzyme responsible for catalyzing the oxidation of hypoxanthine to xanthine which further leads to the formation of uric acid [40] [55] [56]. When it is elevated above normal level, it has been reported to be the underlying reason of gouty arthritis attack and other inflammatory related diseases [55] [56] [57]. Considering the present study, all the analyzed samples exhibited more than ≥50% inhibition against XO activities at 1.00 mg/mL concentration. This signifies that the analyzed compounds effectively inhibited XO from catalyzing the action of converting xanthine to uric acid at a considerably low concentration of 0.1%. Compound 2 was found to exert the highest inhibition capacity at this concentration with 75.63% ± 0.001% inhibitory capacity. It however elicited IC₅₀ at 0.36 mol/L (Table 3).

This was followed by compound 1 (51.03% ± 0.001%) with IC₅₀ 0.52 mol/L. Following closely is compound 4, which elicited an inhibitory capacity of 54.22% ± 0.001% also at this concentration. It however had IC₅₀ of 0.83 mol/L. On the other hand, ligand L exhibited it highest inhibitory capacity of 54.22% ± 0.001% at 1.00 mg/mL with IC₅₀ of 0.85 mol/L. The order of activity for the analyzed compounds is 2 > 1 > 5 > 4 > L > 3. This observed order suggests that, with respect to inhibition of xanthine oxidase, coordination of metal ions to ligand L improved the activity of ligand L, with the exception of compound 3. Therefore, this validates the hypothesis that coordination compounds of biologically active compounds may enhance their activity. The exception of compound 3 in this case indicates that the geometry assumed by the complex and the central metal ion are factors related with the reactivity of coordination compounds [50] [51]. It also reiterates the fact that these factors may be used in fine-tuning coordination compounds towards appropriate usage [58]. The positive control, allopurinol, recorded maximum inhibition of XO activities with 68.36% at 0.025 µg/mL and IC₅₀ of 7.08 mol/L. It had been reported previously that samples causing > 50% XO enzyme inhibition at concentration of 50 µg/L warranted further investigation (Sweeney, 2001) [59]. Although the compounds tested in this research exhibited relatively good activity, they do not fall within this category.

Two important approaches for the design and syntheses of anti-inflammatory agents are based on the inhibition of two enzymes, one of which is lipoxygenase, (LOX). LOX is involved in the biosynthesis of mediators in inflammation. This is because it is involved in the metabolism of arachidonic acid, a precursor to leukotrienes. As a consequence, it is a key target for the development of new anti-inflammatory agents [15] [17] [60] [61] [62] [63]. Results for the lipoxygenase inhibitory activity of ligand L and compounds 1-5 are presented in Table 4. In this case the result obtained points to the fact that ligand L is a better inhibitor of LOX in comparison with the coordination compounds. This therefore suggests that coordination, in case of ligand L, with respect to the metal ions used, may not necessarily improve the LOX inhibitory ability of ligand L. The compounds exhibited a dose dependent activity with the highest activity observed at 1.00 mg/mL. Inhibition levels were within the range of 6.90% - 89.35% within the

concentration of 0.03 - 1.00 mg/mL. The order of the lipoxygenase inhibitory activity of the assayed compounds was L > 1 > 5 > 4 > 3 > 2. Ligand L exhibited the best activity with 89.35% ± 0.001% and IC₅₀ of 0.22 mol/L. It was followed by compound 1 with 71.29% ± 0.001% with IC₅₀ 0.32 mol/L. The least inhibitory activity, at this same concentration, was expressed by compound 2 with 48.70% ± 0.001% and IC₅₀ of 0.96 mol/L. The standard, quercetin, similar with the synthesized compounds, elicited a dose dependent inhibitory activity with maximum activity of 71.50% ± 0.001% at 100 µg/ml. It however, gave an IC₅₀ of 55.91 mol/L.

During inflammation, as part of their defensive roles, leukocytes release their lysosomal enzymes, causing further tissue damage and subsequent inflammation [15] [64]. Damage to cell membranes will further make the cell more susceptible to secondary damage by means of free radical-induced lipid peroxidation [15] [65]. Regulation of the volume and water content of cells may occur, through membrane proteins, by controlling the movement of sodium and potassium ions and damage to the membrane will affect this function [15] [64]. As the red blood cell membrane is similar to that of lysosomal membrane, inhibition of red blood cell hemolysis may provide insights into the inflammatory process [21]. Stabilization of these cell membranes may retard or inhibit the lysis and subsequent release of the cytoplasmic contents which, in turn, minimize the tissue damage and, hence, the inflammatory response [15] [64]. Therefore, substances that contribute significant protection of cell membrane against injurious substances are important in the event of inhibiting the progression of inflammation.

The membrane stabilizing activities of the ligand L and compounds 1-5 are presented in Table 5(a)-(f). The results showed that the activities of the assayed compounds were higher than that of the standard drugs even at lower concentration ranges. This suggests that they are highly potent on human erythrocyte, adequately protecting it against hypotonic induced lyses. All the tested compounds, i.e. ligand L and compounds 1-5, showed dose dependent membrane stabilizing activity over all the concentration ranges. Ligand L exhibited a minimum membrane stability of 38.166% ± 0.27% at 2 mg/mL and maximum activity of 91.3199% ± 1.55%, at 0.20 mg/mL. The mode of response of the erythrocyte was both monophasic and biphasic (Table 5(a)). Similarly, the response of the red blood cells to compound 1 was monophasic and biphasic at lower and higher concentrations of the compound, respectively (Table 5(b)). Compound 1 exerted a minimum membrane stability of 15.27% ± 8.53% at 0.4 mg/mL and maximum activity of 62.51% ± 10.27%, at 1 mg/mL.

Compound 2 also exerted minimum and maximum percentage stability activities of 12.89% ± 3.61% and 75.63% ± 8.40%, at 0.2 and 0.6 mg/mL respectively. The response of the red blood cell was also monophasic and biphasic at all the tested concentrations (Table 5(c)).

At 0.4 and 0.6 mg/mL concentration compound 3 exerted minimal and maximal percentage stability activities of 8.13% ± 0.25% and 67.70% ± 0.78% respectively (Table 5(d)). The response of the red blood cell was also monophasic and biphasic at all the tested concentrations for this compound. For compound 4 the lowest percentage activity was 9.31% ± 2.29% at 0.40. On the other hand, the highest percentage activity was 66.67% ± 0.78% at 1.00 mg/mL concentration (Table 5(e)).

Compound 5 gave membrane stability of 93.40% ± 1.04% and 71.92% ± 8.85% as maximum and minimum percentage activity respectively. The response of the red blood cells was also monophasic and biphasic to this compound (Table 5(f)). It is interesting to note that the ligand L and compounds 1-5 all elicited significantly better membrane stabilizing ability in relation with the standard drug (diclofenac).

The result obtained indicated that ligand L provided the highest protection against induced lyses of the tested cells (Table 5(a)). This therefore suggest that coordination, in this case, did not enhance the membrane stability activity of ligand L. Earlier workers have suggested that one of the possible modes of action of anti-inflammatory drugs involves their binding to the erythrocyte membranes with subsequent alteration of the surface charges of the cells [41] [66] [67]. A consequence of this is that they prevent physical interaction with aggregating agents. It is therefore, suggested that this may be the reason behind the better activity of ligand L in comparison to its coordination compound counterparts. Other researchers further suggested that anti-inflammatory drugs may promote

dispersal of like charges which are involved in the haemolysis of red blood cells, by mutual repulsion [41]. This may also be the case for ligand L, which is purely organic in nature. Buttressing this fact are reports that allude to the fact that certain phenolic compounds exert profound stabilizing effect on lysosomal membrane both in vivo and in vitro [41] [66] [67] . In addition to this, some phenolic compounds have been reported to bind to cations and other macromolecules on the membrane, forming insoluble complexes thereby stabilizing erythrocyte membranes [41] [66] [67]. In this research, our investigation revealed that both ligand L and compounds 1-5 were able to prevent lysis of the tested erythrocyte membranes significantly. This therefore, suggests the potential of these compounds as good anti-inflammatory agents. The observed activity may be attributable in part to the phenolic substituent and the ability of these compounds to bind with ions and molecules on the erythrocyte membrane. The result obtained may therefore stand to further validate, the aforementioned mechanisms of activity.

The fact that denaturation of protein molecules is linked with inflammation process in conditions like arthritis is well documented in literature [15]. In fact, according to Gunathilake et al., 2018, one of the main mechanisms of action of non-steroidal anti-inflammatory drugs, NSAIDs, in the market, is the protection against protein denaturation [15]. The results obtained demonstrated that ligand L and compounds 1-5 can significantly and dose dependently protect against protein denaturation. Compound 2 exhibited a significantly higher (p ≤ 0.001) level of inhibition compared to other compounds studied with percentage activity of 63.38% ± 0.001% at 1.00 mg/mL and IC₅₀ of 0.47 mol/L (Table 6). Whereas compound 5 showed the lowest inhibition levels with 42.08% ± 0.001% also at 1.00 mg/mL with IC₅₀ 1.41 mol/L. The order of the inhibition by the compounds is 2 > 3 > 1 > L > 4 > 5. The result obtained from this investigation suggested good ability of the tested samples in small concentrations to protect against protein denaturation (Table 6). However, the standard drug, diclofenac, at all tested concentration, depicted significantly (p ≤ 0.001) better anti-denaturation activity compared to all the synthesized compounds. The standard drug exhibited its highest activity at 70.04% ± 0.001% at 10 µg /mL with IC₅₀ 1.59 mol/L.

Inflammation is primarily a bodily response to injury, infection or destruction disturbed physiological functions, which entails various processes [15]. Previous researches suggested that most of the non-steroidal drugs act as an anti-inflammatory agent either by inhibiting hydrolytic enzymes, denaturation of proteins or by membrane stabilization process [15]. It may therefore be deduced from the results obtained that chemically and structurally the synthesized compounds are better suited as anti-inflammatory agents by preventing or retarding the rupturing of lysosomal membrane. One of the fundamental goals of this research was to enable us to streamline and put in perspective the biological activity of the synthesized compounds. This is geared toward tailoring the anti-inflammatory activity. In this respect, further definitive studies are necessary, this with further structural analysis for the compounds are currently underway.