Synthesis and Physicochemical Studies for Novel Bioactive Metal Complexes of Macrolide Antibiotic Tylosin

Alaa E. Ali¹, Gehan S. Elasala¹, Yasser S. Mahrous¹, Mohamed H. Sharaf², Ismael M. Elkamhawy^1*

¹Chemistry Department, Faculty of Science, Damanhour University, Damanhour, Egypt.
²Botany and Microbiology Department, Faculty of Science, Al-Azhar University, Cairo, Egypt.

DOI: 10.4236/msa.2022.1310033                

Abstract

Tylosin is a well-established antibiotic that has been widely employed in human and veterinary medicines. It can act as a potential ligand binding metal ions due to various donor atoms in the structure. Our study on the complexation of various metal ions with tylosin ligand revealed that they preferably coordinate with mycaminose fragment to establish Novel trends complexes. Tylosin ligand (TYS) behaves as bidentate for complexation with different metal ions such as Cr(III), Mn(II), Fe(III), Co(II), Ni(II), Cu(II) and Zn(II). Various essential metal complexes of tylosin were synthesized and characterized by techniques such as UV, IR, Elemental analysis, magnetic susceptibility and ESR spectra of Cu(II) complex. These techniques are used to know their geometries and mode of bonding, with stoichiometry, 2:2 (M:L). Thermal analysis (TGA and DTA) of ligands and their metal complexes were carried out to distinguish between the coordinate and hydrate solvents and to estimate the stability ranges, peak temperatures. The thermodynamic parameters, such as activation energy (ΔE^*), the enthalpy of activation (ΔH^*), entropy of activation (ΔS^*) and Gibbs free energy (ΔG^*) are calculated and discussed. Some tylosin complexes show higher activity than tylosin for some bacterial and fungal strains. Low concentration value of minimum inhibitory concentration (MIC) results is 15.625 μg/ml for both complexes [Zn₂(TYS)₂Cl₂(H₂O)₄]·25H₂O and [Cu₂(TYS)₂Cl₂(H₂O)₄]·25H₂O with B. cereus genus maybe a valuable data used to produce novel therapeutic agent. This study constitutes several essential aspects for future research on tylosin metal complexes as antibacterial assessment and as potential medicinal agents.

Keywords

Tylosin Complexes, Synthesis, Spectral, Tetragonal Distortion, Biological Activity

1. Introduction

Tylosin (TYS), a macrolide antibiotic, is produced naturally by the Actinomycetota species Streptomyces fradiae, which was firstly identified by McGuire in 1961 [1]. Tylosin (Figure 1) is a 16-membered branched lactone, tylonolide, with disaccharide (α-L-mycarosylβ-D-mycaminosyl) and monosaccharide (α-D-mycinose) substituents. Tylosin (TYS) mostly consists of tylosin (factor A), but it may also contain small amounts of desmycosin (factor B), macrocin (factor C), and relomycin (factor D) and tylosin (A) being at least 80% of the composition [2] [3]. Tylosin (TYS) has significant commercial importance which is used effectively as a veterinary medication and feed additive to stimulate young animals’ growth. It is highly effective against both Gram-positive and Gram-negative bacteria as well as Gram-positive mycoplasmas. Tylosin is typically reported to be less active against most bacteria, with the exception of Mycoplasma species, as compared to erythromycin (a prototypical macrolide molecule). In ruminants, pigs, and poultry, antibiotic is used to treat a variety of illnesses brought on by sensitive organisms [4] [5]. The wide use of tylosin antibiotic from the macrolide series makes it crucial to regulate their concentration in both dosage forms and biological liquids [6]. Currently, techniques like liquid chromatography [7], high performance liquid chromatography (HLPC) [8] [9] [10] and spectrophotometric methods [11] are employed to identify macrolide antibiotics. Tylosin (TYS) is White to buff-colored powder with a mild musty odor; its melting point is “128˚C - 132˚C” and its solubility in water 5 mg/mL at 25˚C and Freely soluble in methanol. Soluble in lower alcohols, esters and ketones, chloroform, dilute mineral acids, amyl acetate, chlorinated hydrocarbons, benzene, and ether. Tylosin is unstable in acidic and alkaline medium and relatively stable under neutral pH conditions (pH = 7). It is chemically stable under standard ambient conditions (room temperature) [12]. Antibiotic resistance is growing at an alarming

Figure 1. The chemical structure of tylosin A (TYS).

rate and consequently the activity of antibiotics against Gram-positive and Gram-negative bacteria is becoming less effective gradually day by day. In a concrete sense, there is a considerable need for the design of novel antibiotics therapeutic with a good spectrum of activity, so the current study framework is to establish a foundation for antimicrobials.

2. Experimental

Tylosin was dissolved in distilled water, while metal chlorides of Cr(III), Mn(II), Fe(III), Co(II), Ni(II), Cu(II), and Zn(II) were dissolved in ethanol 70%, then several molar ratios were tested until reach to the stoichiometry of (M:L). 40 ml of dissolved heated ethanol solution of chlorides inorganic salts of transition metal ions mixed with water soluble tylosin ligand. The reaction mixture was refluxed for approximately 60 minutes and then allowed to sit overnight. The high yield precipitated complexes were determined to identify the molar ratio of the predicted amount of ligand and the molar amount of the metal chloride salt interacted. The synthesized complexes were then filtered, washed several times with a 70% ethanol solution, and dried in a vacuum desiccator over anhydrous CaCl₂.

3. Measurements

3.1. Elemental Analysis and Physical Measurements

The metal contents were determined by atomic absorption spectrophotometer. Carbon, hydrogen and nitrogen analysis of tylosin and all complexes measured at central lab, Cairo University contents of all the synthesized complexes were analyzed by the usual method. The well-known Volhard method [13] was used in an acidic medium to analyze the complexes’ chloride contents. Melting points were measured by using the FALC melting point device.

3.2. Spectral Studies

The infrared spectra of tylosin and its metal complexes were obtained in a KBr disc using The Bruker Tensor 37 FT-IR instrument which is located in the central lab, Alexandria university, covering frequency range of 400 - 4000 cm⁻¹ and the data recorded and refined by OPUS Data Collection Program.

Electronic spectra of the colored complexes were detected in nujol mull spectra following the method described by Lee, Griswold and Kleinberg [14] using Perkin Elmer lambda 25 uv/vis spectrophotometer, made in England that can Detect the wavelengths between 190 - 1100 nm.

ESR spectra of Cu(II) tylosin complex was measured on an ERS-220 X-band spectrometer (9.45 GHz) using 100-kHz field modulation at room temperature and at 77˚K; g factors were determined in relation to the reference marker DPPH (g = 2.0036).

Magnetic susceptibility measurements were measured at room temperature on a Johnson Matthey magnetic susceptibility balance using Gouy method. Diamagnetic corrections were calculated using Pascal’s constants [15]; the calibrant used was Hg[Co(SCN)₄]. The values of effective magnetic moments were calculated from the following equation ${\mu }_{\text{eff}}=\text{2}.\text{84}{\left(X_M^{corrt}T\right)}^{1/2}$, where, $X_M^{corrt}$ is the molar magnetic susceptibility corrected for diamagnetism of all atoms in the compounds.

3.3. Thermo Gravimetric Analysis (TG) and Differential Thermal Analysis (DTA)

Differential thermal analysis (DTA) and thermo gravimetric analysis (TG) of tylosin and its metal complexes were carried out using the instrument LINSEIS PT1600. The rate of heating was 10˚ K/min. The cell used was platinum and the atmospheric nitrogen rate flow was 15 ml/min.

3.4. Biological Studies

The antimicrobial activity of the tested compounds were determined by means of paper disk diffusion method (7 mm) on Muller Hinton agar for bacteria and potato dextrose agar for Candida [16]. The bacterial indicators were S. aureus (ATCC6538), B. cereus (ATCC10987), for Gram-positive bacteria, E. coli (ATCC 8739), S. Typhi (ATCC14028) as Gram-negative bacteria and one fungal strain C. albicans (ATCC10231).

The minimum inhibitory concentration (MIC) carried out by preparing Different concentrations of compounds. Test sample (100 µl) of various concentrations was added into sterile microtiter plate wells filled with 100 µl of double strength Mueller Hinton (MH) broth to make final concentrations at 1000, 500, 250, 125, 62.5, 31.25. and 15.75 µg/ml. Bacterial cell suspension (50 µl) corresponding to (OD equivalent to 0.5 McFarland standard) was added in all wells except those in the negative control well. Positive Control wells were filled with MH broth and bacterial suspension to check for adequacy of the broth to support bacteria growth [17]. The negative control wells consisted of sterile distilled water and Mueller Hinton broth to check sterility. The plates were incubated at 37˚C for 24 hrs. To indicate bacterial growth, 30 µl of resazurin solution (0.02% w/v) (HiMedia) was added to each well, and the plate was re-incubated overnight. A change in color from blue to purple, red or pink, indicated the growth of bacteria. A change in the color of growth control wells to pink, red or purple indicated the proper growth of the isolate and no change in the color of the sterile control well indicated the absence of contaminants. The experiment was performed in duplicates and mean values were calculated [18].

4. Results and Discussion

4.1. Elemental Analysis and Stoichiometry of Novel Tylosin Metal Complexes

The proportion of elements in the innovative metal complexes was determined and quantified by elemental analysis, Table 1; also certain characteristics such as color, Yield and chemical formula of tylosin macrolide ligand and its metal

Table 1. Indicated elemental analysis, molecular formula, stoichiometry’s, yield and color of tylosin ligand and its metal complexes.

complexes are included. The analytical findings proved that the tylosin ligand reacts in 2:2 (M:L) molar ratio with Mn(II), Co(II), Ni(II), Cu(II), Zn(II), Fe(III) and Cr(III). The complexes are highly hydroscopic. All the complexes are slightly soluble in N,N-dimethylformamide (DMF) and highly soluble in dimethyl sulfoxide (DMSO-d6). While, in ethanol, methanol and water, all complexes are insoluble. All of the complexes had a melting point values greater than 325˚C.

4.2. Infrared (IR) Spectra of Tylosin and Its Metal Complexes

The infrared spectrum of tylosin was interpreted by correlating the functional groups it contains in the structure of molecule to the spectrum it produces [19] [20] [21]. Spectroscopic characteristics of tylosin (Figure 2) revealed an absorption band which is observed at 1720 cm⁻¹ assigned to the lactone carbonyl group. The absorption bands at 1460, 1510 and 1560 cm⁻¹ are associated with stretches vibrations of C=O and C=C. There are several absorption bands in the range of 1050 to 1300 cm⁻¹ that are associated with the C-O group of the alcohol, ether, and ester functionalities. The C-N group promotes an absorption band in the 1180 - 1280 cm⁻¹ range. The C=O group is connected to the bands of absorption that are related to the functions of ester, aldehyde and ketone at 1380 cm⁻¹, 1560 cm⁻¹, and between 1650 and 1750 cm⁻¹. The band around 1420 cm⁻¹ is attributed

Figure 2. Infrared spectra of tylosin (TYS) ligand and its metal complexes.

to the angular deformation of CH₂ beside carbonyl. In the 1620 - 1680 cm⁻¹ range, there are absorption bands related to C=C deformation. The absorption bands in the range of 2700 - 2960 cm⁻¹ are related to the C-H group. Absorption bands in the region of 670 to 970 cm⁻¹ are related to the CH=CH group. The OH group is responsible for the absorption band that spans the wavelengths of 3200 to 3600 cm⁻¹.

The infrared spectrum of tylosin metal complexes for Zn²⁺, Cu²⁺, Mn²⁺, Co²⁺, Ni²⁺, Cr³⁺ and Fe³⁺ were recorded and then Compared to the IR spectrum of TYS ligand. The bands in the range (1720 - 1735) cm⁻¹ were also noticed in the tylosin metal complexes spectral sheet, which was assigned to the lactone carbonyl group which is strong evidence that there is no coordination between the lactone carbonyl group and the metal ions. The main differences are changes in the relative intensity for OH group (3200 to 3600) cm⁻¹, which can conclude that metal ions coordinated with poly hydroxyl mycaminose fragment. The band at 1250 cm⁻¹, which assigned to C-N group in ligand spectral sheet, has been red shifted to the range 1125 - 1163 cm⁻¹ which indicates that the metal ions may be bind with 3˚ amine donor group to form a stable metal complexes structure.

In the far IR spectra, the presence of M-N stretching vibration in the (510 - 585) cm⁻¹ range which is absent in the free ligand provides evidence that moiety is bonded to the metal ion through nitrogen. Also the new bands were detected in the range (440 - 485) cm⁻¹ detected in the complexes assigned to M-O which confirmed the bonding of nitrogen and oxygen to metal ions.

4.3. Electronic Absorption Spectra and Magnetic Susceptibility Studies

Generally, the magnetic moment readings for metal ions at room temperature as well as the Nujol mull electronic spectral data of synthesized metal complexes have been applied to determine the possible geometry of these synthetic complexes. These measurements have been clarified in Table 2. However; each metal ion complex will be described and discussed independently as follows.

4.3.1. Nickel Complex

The values of µ_eff of Ni(II) complex [Ni₂(TYS)₂(Cl)₂(H₂O)₄], at room temperature were found to be 3.20 BM which is higher than of µ_s.o value that is characterized for two unpaired electrons of Ni(II) octahedral complexes. The ensuing high value of magnetic moment may be a sign of Strong spin orbit coupling under which µ_eff = µ_S.O.(1 − 4λ/10Dq). As the spin orbit coupling constant λ for d⁸ system is (λ = −315), µ_eff becomes greater than µ_S.O. Ni(II)-TYS complex shows four bands in their electronic spectra, suggesting distorted tetragonal geometry (D_4h). These bands can be assigned to the following transitions [22].

${\nu }_{\text{1}}={}^3\text{B}{}_{\text{1g}}\to {}^3\text{E}{}_{\text{g}}$ 10Dq – 35/4Dt

${\nu }_{\text{2}}={}^3\text{B}{}_{\text{1g}}\to {}^3\text{B}{}_{\text{2g}}$ 10Dq

${\nu }_{\text{3}}={}^3\text{B}{}_{\text{1g}}\to {}^3\text{A}{}_{\text{2g}}$ 18Dq – 4Ds – 5Dt

${\nu }_{\text{4}}={}^3\text{B}{}_{\text{1g}}\to {}^4\text{E}{}_{\text{g}}$ 18Dq – 2Ds – 25/4Dt

The tetragonal character is measured by the transition ³B_1g→³E_g^(a). The transition ³B_1g→³B_2g^(b), on the other hand, is unaffected by Ds and Dt while is effectively the measure of the infield splitting parameter Dq^xy. The axial distortion Dt is computed by using the Wentworth and Piper formula [23], which is Dt = 4/7(Dq^xy − Dq^Z) or by another derived formula 35/4Dt = ν₂ − ν₁. The equation 2ν₁ − ν₂/10 was used to compute Dq^Z. The zero field splitting parameter (D) has also been calculated using the expression D = 9k₁/α²[(µ_eff/µ_s.o)² − (µ_eff/µ_s.o) + 1S] while k₁, is the splitting of the initial excited state, and it is given as k₁ = 35/4Dt. The values for the various parameters are listed in Table 2. The presence of four bands indicates a significant tetragonal deviation from octahedral symmetry. The positive value of Dt, Ds and smaller value of Dq^z than Dq^xy are assigned to tetragonal elongated octahedral geometry of Ni(II) complex. The features of all produced bands that represent these complex transitions as shown in Figure 3 were in strong feature and well analyzed; the complex Ni(II)-TYS shows four bands at 1041.7, 941.6, 541.1 and 416.7 nm, which correspond to the transitions ³B_1g → ³E_g (υ₁), ³B_1g → ³B_2g (υ₂), ³B_1g → ³A_2g (υ₃) and ³B_1g → ⁴E_g (υ₄) respectively, that is a great elucidation of tetragonal elongated octahedral structure.

Table 2. Indicates Nujol mull electronic absorption spectra (nm·cm⁻¹), room temperature magnetic moment values (µ_eff, 298˚K) B.M, Ds, Dt, Dq^z and Dq^xy of Tylosin Metal complexes.

Figure 3. Showed electronic absorption spectra of different metal complexes of Tylosin.

4.3.2. Cobalt Complex

The expected calculated value of the spin only magnetic moment (µ_s.o)of Co(II) is 3.87 B.M due to Co(II) is a d⁷ system with three unpaired electrons, while the actual values of magnetic moment for complex [Co₂(TYS)₂(Cl)₂(H₂O)₄] is 4.79 B.M respectively. The irregular values for Co(II)-TYS complex µ_eff may be attributed to ⁴T_1g ground state crystal field term in octahedral symmetry which makes a significant contribution to the magnetic moment of Co(II) complex in octahedral symmetry [24]. Typically, Three absorption bands are predicted to occur in the electronic spectra of Co(II) complex with perfect high spin octahedral geometry due to three probable spin allowed transitions, ⁴T₁g(F) → ⁴T₂g(F), ⁴T₁g(F) → ⁴T₁g(P) and ⁴T₁g(F) → ⁴A₂g(F) that represent (ν₁), (ν₂) and (ν₃) respectively, while due to the splitting of ⁴T_1g(F), ⁴T₂g and ⁴T_1g(P) in their electronic spectra, these cobalt complex of tylosin display four bands in their electronic spectra, as shown in (Figure 3), which are an evidence of axially distorted octahedral symmetry around Co(II) in these metal complexes. The electronic spectra of Co(II)-TYS complex reveal four bands, indicating axially distorted octahedral symmetry around Co(II). These bands can be ascribed to the transitions mentioned below under D_4h symmetry [24]:

${\nu }_{\text{1}}={}^4\text{A}{}_{\text{2g}}\to {}^4\text{E}{{}_{\text{g}}}^{(}{}^{\text{a})}$ 6/5Ds – 15/4Dt

${\nu }_{\text{2}}={}^4\text{A}{}_{\text{2g}}\to {}^4\text{B}{}_{\text{2g}}$ 8Dq + 4/5Ds – 13Dt

${\nu }_{\text{3}}={}^4\text{A}{}_{\text{2g}}\to {}^4\text{E}{{}_{\text{g}}}^{(}{}^{\text{b})}$ 8Dq + 4/5Ds – 17/4Dt

${\nu }_{\text{4}}={}^4\text{A}{}_{\text{2g}}\to {}^4\text{B}{}_{\text{1g}}$ 18Dq + 4/5Ds – 13Dt

${\nu }_{\text{5}}={}^4\text{A}{}_{\text{2g}}\to {}^4\text{E}{{}_{\text{g}}}^{(}{}^{\text{c})}$ 6Dq + 2/5Ds – 6Dt

${\nu }_{\text{6}}={}^4\text{A}{}_{\text{2g}}\to {}^4\text{A}{{}_{\text{2g}}}^{(}{}^{\text{a})}\left(\text{P}\right)$ 6Dq+ 18/5Ds – 6Dt

The value of Dq^(xy) has been calculated by using the derived equation $\frac{{\nu }_4-{\nu }_2}{10}$ and D_t value has been estimated from the equation $\frac{4}{35}\left({\nu }_3-{\nu }_2\right)$. The common equation, Dt = 4/7(Dq^xy − Dq^z) used to deduce the value of Dq^z. The positive value of Dt and Ds produced mathematically are the indication of tetragonal elongated octahedral geometry of Co(II) complex . The smaller value of Dq^z, than Dq^xy, support and clarify the tetragonal elongation along z-axis in Co(II) complex of tylosin. The spectra for Co(II)-TYS complex shows definitely four bands at 1073, 920, 494 and 402.4 nm that correspond to the transitions (ν₂) ⁴A_2g → ⁴B_2g, (ν₃) ⁴A_2g → ⁴E_g^(b), (ν₄) ⁴A_2g → ⁴B_1g and (ν₅) ⁴A_2g→⁴E_g^(c) ,respectively. The last band n₆ that represents the transition ⁴A_2g → ⁴A_2g^(a) (P) which may be confused with the final band appeared in the spectra. The band ν₁ refers to ⁴A_2g → ⁴E_g^(a) transition state that is unnoticeable due to the small gap between the two levels which causes absorption energy in the infrared range. From D_s and D_t values, the value of ν₁ has been estimated and found in 3486 cm⁻¹, which refers to the infrared spectrum.

4.3.3. Chromium Complex

The magnetic moment of Cr(III) complex of tylosin, at room temperature was found 3.74 B.M, as shown in Table 2, this value of Cr(III) metal complex is close to spin only value that equal 3.87 B.M thereby, suggesting an octahedral geometry around the d³ system of chromium ion. The different values of µ_s.o and µ_eff may be an indication of distorted octahedral geometry of this complex [25]. The octahedral geometry of Cr(III)-TYS complex is determined due to the presence of three bands that represent the three lowest spin-allowed transitions ⁴A_2g → ⁴T_2g(F), ⁴A_2g → ⁴T_1g(F) and ⁴A_2g → ⁴T_1g(p), while four bands appeared in distorted octahedral geometry due to splitting occurred for ⁴T_2g to ⁴E_g^a and ⁴B_2g and also scattering of ⁴T_1g(F) to ⁴A_2g^a and ⁴E_g^b [26]. These four bands that represent distorted tetragonal octahedral geometry can be assigned to the following transitions.

${\nu }_{\text{1}}={}^4\text{B}{}_{\text{1g}}\to {}^4\text{E}{{}_{\text{g}}}^{\text{a}}$ 10Dq – 35/4Dt

${\nu }_{\text{2}}={}^4\text{B}{}_{\text{1g}}\to {}^4\text{B}{}_{\text{2g}}$ 10Dq

${\nu }_{\text{3}}={}^4\text{B}{}_{\text{1g}}\to {}^4\text{A}{{}_{\text{2g}}}^{\text{a}}$ 18Dq – 4Ds – 5Dt

${\nu }_{\text{4}}={}^4\text{B}{}_{\text{1g}}\to {}^4\text{E}{{}_{\text{g}}}^{\text{b}}$ 18Dq – 2Ds – 25/4Dt

The calculated value of Dq^xy is estimated from the transition ν₂ that is equal to 10Dq^xy. The value of Dt is calculated by the difference between ν₁ and ν₂ which is equivalent to (35/4)Dt. The symmetry plane of Dt is related to the in-plane and out-of-plane field strengths as Dt = 4/7(Dq^xy − Dq^Z) which shows that Dq is in-plane (xy) and out-of-plane (z) Field strength, respectively. The parameter Ds has been calculated from the equation (ν₃ − ν₂) = 8Dq − 4Ds − 5Dt. The presence of four bands in the electronic spectral sheet of dark green chromium (III) metal complex in the range between (350 - 550) nm is a great indication for distorted octahedral geometry. The positive value of the two field parameters Ds and Dt proved this distorted geometry. The smaller value of Dq^xy than Dq^z show axially elongated tetragonal structure for these metal complexes.

4.3.4. Copper Complex

Cu(II) complex of tylosin has shown a magnetic moment near 2.17 B.M, which corresponds to the presence of a single unpaired electron due to d⁹ system of cupric ion that indicates mononuclear nature of the complex with mild paramagnetic. The difference between the spin only and magnetic moment values may be due to Strong spin orbit coupling which allows a greater value of µ_eff than µ_S.O. For copper (II) metal complex of tylosin, the coordination number was six. It means that the geometry of these complexes may be distorted octahedral as tetragonal elongated structure, which can clearly determine the geometry by illustrate the electronic spectra of these Cu(II) metal complexes. In the pure octahedral coordination geometry, the ²D state of Cu(II) is split into ²E_g ground state and ²T_2g excited state with a single electronic transition. Therefore, Cu(II) is no longer in the pure octahedral (Oh) geometry, and the symmetry around the metal is slightly distorted. A Jahn-Teller (J-T) distortion is taking place that changes the symmetry from octahedral (O_h) to tetragonal structure (D_4h) [27]. Due to the Jahn-Teller effect, the ground state ²E_g is further scattered into ²B_1g ground and ²A_1g excited states, and the transition state ²T_2g is separated into ²B_2g ground and ²E_g excited states. Therefore, the three bands that indicate distorted tetragonal octahedral geometry can be described by the following transitions [28]:

${\nu }_{\text{1}}={}^2\text{B}{}_{\text{1g}}\to {}^2\text{A}{}_{\text{1g}}$ 4Ds + 5Dt

${\nu }_{\text{2}}={}^2\text{B}{}_{\text{1g}}\to {}^2\text{B}{}_{\text{2g}}$ 10Dq

${\nu }_{\text{3}}={}^2\text{B}{}_{\text{1g}}\to {}^2\text{E}{}_{\text{g}}$ 10Dq + 3Ds – 5Dt

For Cu(II)-TYS complex, the electronic spectral exhibits clearly three bands at 9770, 16100 and 17200 cm⁻¹ which can be attributed to three optical probable transitions, ²B_1g → ²A_1g (ν₁), ²B_1g → ²B_2g (ν₂) and ²B_1g → ²E_g(ν₃). In the spectrum, there is a scattered broad band at range 580 - 625 nm that reveals scattering of ²T_2g of octahedral geometry into ²B_2g and ²E_g which are close to each other [29], while the first band appeared lately at 1023 nm which assigned to ²B_1g → ²A_1g transition that proved distortion of axially tetragonal elongation for Cu(II) Tylosin metal complex. The value of 10Dq is estimated by determination of the second band (ν₂) at which the value of Dq^z by cm⁻¹ is calculated by division of (ν₂/10). The value of Dq^xy is calculated directly from the popular equation that equal Dt = 4/7(Dq^xy − Dq^Z). The tetragonal crystal field parameters Ds and Dt founded by solving the following two equations, ν₃ = 4Ds + 5Dt → (1) and ν₂ – ν₁ = −3Ds + 5Dt → (2). The same positive charge value for both parameters Ds and Dt indicated an axially elongated tetragonal geometry for both structure of Cu(II) metal complexes.

4.3.5. Iron Complex

Magnetic moments of Fe(III) metal complex was founded to be 5.60 B.M; this value is quite near to the spin-only magnetic moment corresponding to five unpaired electrons. This is due to the fact that both Fe(III) metal complexes have a high magnetic spin and are dilute octahedral complexes. Spin-orbit coupling, which limits the spin and orbital motion of electrons to an extent, could explain the somewhat lower magnetic moment value. In the electronic spectral sheet of Fe(III)-TYS complex, there were four weak bands were detected as listed in Table 2 which attributed to the following spin forbidden transitions [30],

${\nu }_{\text{1}}={}^6\text{A}{}_{\text{1g}}\to {}^4\text{T}{}_{\text{1g}}\left(\text{4G}\right)$,

${\nu }_{\text{2}}={}^6\text{A}{}_{\text{1g}}\to {}^4\text{T}{}_{\text{2g}}\text{or}{}^{\text{4}}{\text{A}}_{\text{1g}}\text{or}{}^{\text{4}}{\text{E}}_{\text{g}}\left(\text{4G}\right)$,

${\nu }_{\text{3}}={}^6\text{A}{}_{\text{1g}}\to {}^4\text{E}{}_{\text{g}}\left(\text{4D}\right)$,

${\nu }_{\text{4}}={}^6\text{A}{}_{\text{1g}}\to {}^4\text{T}{}_{\text{1g}}\left(\text{4P}\right)$.

The presence of these four weak bands is indicative of axial elongated distorted octahedral geometry for these Fe(III) complexes. So, the values of different crystal field parameters have not been estimated due to the high spin nature of Fe(III) metal. The suggested geometry of the reddish brown Fe(III)-TYS complex depends on a bidentate nature of ligand, chlorides and water which allow six coordination numbers around Fe(III) that confirm distorted octahedral structure. The first bands in the UV/Vis spectra of Fe(III) which exhibits in the region range 300 - 350 nm due to ligand metal charge transfer.

4.3.6. Manganese Complex

Magnetic moments of Mn(II)-TYS complex was measured to be 5.75 B.M; the spin only magnetic moment corresponding to five unpaired electrons is very close to this value, which is attributable to the fact that Mn(II) metal complex is dilute octahedral with a high magnetic spin. The slightly low magnetic moment value could be due to spin-orbit coupling, which limits the spin and orbital motion of electrons to a small extent. The Mn(II) complex of tylosin exhibit four very weak bands that was recognized in its electronic spectral data at 18,060, 24,410, 26,380 and 27,610 cm⁻¹ assigned to the following spin forbidden transitions [31], ν₁ = ⁶A_1g → ⁴T_1g (4G), ν₂ = ⁶A_1g → ⁴T_2g or ⁴A_1g or ⁴E_g(4G), ν₃ = ⁶A_1g → ⁴E_g (4D) and ν₄ = ⁶A_1g → ⁴T_1g (4P). The presence of these weak bands is indicative of axial elongated distorted octahedral geometry for these Mn(II) complex. So, the suggested geometry of Mn(II)-TYS complex depends on a bidentate nature of ligand, chlorides and water which allow six coordination numbers around Mn(II) that confirm distorted octahedral structure.

4.3.7. Zinc Complex

For [Zn₂(TYS)₂(H₂O)₄Cl₂] complex, the proposed structure due to the bidentate nature of Tylosin through oxygen atoms of OH group of mycarose sugar and Nitrogen atom of 3˚ amine group in the structure with the presence of two chloride ions and four water molecules in the inner sphere that may be a good conclusion for octahedral geometry of this complex. Only a high intensity band at 329 - 365 nm was observed in this complex, which was attributed to ligand metal charge transfer. No d-d transition could be observed due to the d¹⁰ structure of Zn(II); hence the stereochemistry could not be deduced from ultraviolet and visible spectra. However, distorted octahedral geometry is proposed for this complex based on comparisons between their spectra and those of related environments.

4.4. Electron Spin Resonance (ESR) of Cu(II)-TYS Complex

In the [Cu₂(TYS)₂Cl₂(H₂O)₄]·25H₂O, we have observed the relation $g_{\left|\right|}>g_{\perp }>g_e$ and the observed $g_{\left|\right|}$ and $g_{\perp }$ values which tended to be 2.43 and 2.10 ,respectively. These values are feature of Cu(II) ions coordinated by six ligands which form a distorted octahedral elongated along the z-axis. The ground state of the paramagnetic electron is ²B_1g (d_x2-y2 state). Moreover, copper has nuclear spin 3/2 which couples with the electron spin to form four lines hyperfine splitting of the EPR spectrum (Figure 4). The nature of ligand-metal bond can ascribe according to the calculated values of α², ${\beta }_1^2$ and β², Table 3 indicates the covalent character of the bond. The G value is founded to be 4.5 which confirms the negligible exchange coupling takes place. The value of G can be correlated by the room temperature magnetic moment value that equals 2.17 B.M for this complex, higher than the value signs to one unpaired electron 1.73 B.M due to the orbital contribution in the spin of the complexes and confirmed that the exchange interaction is negligible. Electronic absorption spectra of [Cu₂(TYS)₂Cl₂(H₂O)₄]·25H₂O showed the absorption bands E₁(²B_1g → ²B_2g) and

Figure 4. Showing ESR spectra of Cu(II)-TYS complex.

Table 3. Room temperature ESR spectral parameters for copper (II) complex of Tylosin.

E₂(²B_1g → ²E_g) that attributed to the values 16,100 and 17,200 cm⁻¹, respectively. By comparing these values by the produced one from EPR data sheet calculations showed slightly different values as follows E₁(²B_1g → ²B_2g) = 15,680 cm⁻¹ and E₂(²B_1g → ²E_g) = 17,604 cm⁻¹. This correlation shows a great matching between the results of both electronic absorption spectra and EPR spectrum [32] [33] [34].

From elemental analysis, IR, electronic absorption spectra and magnetic moment values concluded structures shown in (Figure 5 and Figure 6).

4.5. Thermal Analysis

The current thermal analysis study aims to learn more about the effect of temperature on metal complexes. With a heating rate of 10˚C per minute, TG/DTA analytical techniques were used to extensively study the thermal stability of the complexes. Their decline profiles are observed at different temperature ranges,

Figure 5. Proposed structure of M(II)-TYS complexes, where M = Mn(II), Co(II), Ni(II), Cu(II) or Zn(II).

Figure 6. Proposed structure of M(III)-TYS complexes, Where M = Cr(III) or Fe(III).

resulting in a variety of thermally stable products [35] [36]. The DTA/TGA signal data, Figure 7 for the TYS ligand dedicated two peaks. The first was endothermic and the other one was exothermic with T_max near 83.6 and 512.4˚C and their activation energies were 20.109 and 19.226 kJ/mole, respectively. The decomposition orders are 1.58 and 0.89 which confirms that the order types are second and first, respectively. The TGA data included clearly two major steps which showed great matching with DTA data where the first step was due to the dehydration of the outer water sphere. While the other one due to the destruction of the partially dehydrated ligand with the main exit of NO₂ as a result of TYS thermal cracking. The TGA sheet indicated that TYS ligand destructed without any residue percentage, as shown in Scheme 1.

Both of [Cr₂(TYS)₂Cl₄(H₂O)₂]·27H₂O and [Ni₂(TYS)₂(Cl)₂(H₂O)₄]·28.8H₂O complexes are hydroscopic due to large number of outer water molecules as evidenced by a significant decline on TGA curve for both Figure 8 and Figure 9. The DTA given data For the complex [Cr₂(TYS)₂Cl₄(H₂O)₂]·27H₂O at heating

Figure 7. TG/DTA curves of TYS ligand.

Figure 8. TG/DTA curves of [Cr₂(TYS)₂Cl₄(H₂O)₂]·27H₂O complex.

Figure 9. TG/DTA curves of [Ni₂(TYS)₂Cl₂(H₂O)₄]·28.8H₂O complex.

rate 10˚C/min showed three peaks one endothermic at 116.0˚C and the others were exothermic with T_max 305.8˚C and 424.2˚C with activation energies 12.198, 32.903 and 92.863 KJ/mole with orders 1.38, 1.02 and 1.71, respectively. All orders are of the first type except the last one is the second type. The TG curve shows a great decline starting from temperature 46.0˚C till 184.1˚C which represents a weight loss 18.71%. This high ratio of weight loss is due to a large number of outer sphere water adsorbed molecules about 27 moles. A large number of adsorbed water in this early temperature range can be explained that this metal complex is hygroscopic type and this result is greatly confirmed with a result of Karl Fischer test. By following up the TG curve noticed the second and third steps of Cr(III) thermal cracking which agreed with TGA profile. The end of thermal cracking for [Cr₂(TYS)₂Cl₄(H₂O)₂]·27H₂O complex is formation of Cr₂O₃ and remain of two carbon atom which represents by residue ratio equal 6.74%. The detailed mechanism of cracking for this complex can be shown in designated Scheme 2. Thermal analysis DTA and TGA curves, Figure 9 for [Ni₂(TYS)₂(Cl)₂(H₂O)₄]·28.8H₂O complex show some variation from the given values of the other metal complex [Cr₂(TYS)₂Cl₄(H₂O)₂]·27H₂O which the thermal cracking for this both complexes occurred through three steps while double decomposition steps for Ni(II)-TYS complex.

The DTA signal data for [Ni₂(TYS)₂(Cl)₂(H2O)₄]·28.8H₂O complex, at a heating rate 10˚C/min gave two peaks one endothermic at T_max 130.0˚C and the second one exothermic at T_max 338.3˚C with activation energies 14.170 and 38.827

Scheme 1. Illustrated thermal cracking of Tylosin (TYS) ligand.

KJ/mole. The orders of the reactions are 1.46 and 1.40 respectively, indicating the first order for both of them. This can be demonstrated by TGA data which Ni(II) complex shows thermal stability till 79.8˚C and produced well defined two peaks at (79.8˚C - 248.0˚C), (248.0˚C - 696.6˚C) with a weight loss of 19.88% which represent the dehydration of this complex and 72.55% that describe the cracking of rest molecule except for the ratio of remained part 7.57%. The process of decomposition of the complex ended with the formation of two moles of metal oxide NiO and 4C. The decomposition technique can be shown in Scheme 3. The values of DTA thermodynamic analysis parameters of Tylosin and its metal complexes such as activation energy (ΔE*), enthalpy of activation (ΔH*), entropy of activation (ΔS*) and Gibbs free energy (ΔG*)calculated and summarized in Table 4.

Scheme 2. Indicates steps of [Cr₂(TYS)₂Cl₄(H₂O)₂]·27H₂O thermal cracking.

4.6. Biological Assessment

4.6.1. Antimicrobial Activity

Using paper disk diffusion method [37], the tylosin ligand and its synthesized metal complexes were evaluated for their antimicrobial activities. Thus, these compounds were screened against Staphylococcus Aureus (S. aureus); Bacillus cereus (B. cereus) as a Gram-positive bacteria, Escherichia Coli (E. coli); Salmonella typhimurium (S. typhi) as Gram-negative bacteria and Candida Albicans (C. albicans)as a fungal strain. The results displayed in Table 5 clearly indicate that the free ligand has exhibited inhibition effect on all tested microorganisms

Scheme 3. Indicates steps of [Ni₂(TYS)₂(Cl)₂(H₂O)₄]·28.8H₂O thermal cracking.

due to Structure-activity relationships (SAR) of tylosin that was evaluated in terms of their antimicrobial and ribosome-binding activities blocking the synthesis of protein [38].

However, the [Zn₂(TYS)₂Cl₂(H₂O)₄]·25H₂O and [Cu₂(TYS)₂Cl₂(H₂O)₄]·25H₂O tested compounds exhibited potent antimicrobial effect especially for Gram-positive bacterial strains (S. aureus); (B. cereus) and fungal strain (C. albicans) in comparison to the reference free ligand standard tylosin [38]. The

Table 4. DTA analysis parameters of Tylosin and its metal complexes.

Maximum of inhibition activity (38 mm) was observed against (S. aureus) by Cu(II)-TYS complex. The enhanced microbial activity of zinc and copper complexes with tylosin ligand may be illustrated using chelation effect theory [39].

4.6.2. Minimum Inhibitory Concentration (MIC)

MIC is useful in research facilities because it may be used to monitor antibiotic resistance and to track the activity of new antimicrobial drugs [40]. MIC is applied for some of tylosin-Metal complexes that have highest antimicrobial activity results against tested species reported in Table 5, MIC results of ligand and its metal complexes by µg/ml are summarized in Table 6.

Table 5. Biological assessment expressed by (10 mg/ml) for free ligand tylosin and tested complexes againstbacterial strains and Candida albicans.

Table 6. Illustrate Minimum inhibitory concentration (MIC) by (µg/ml) of tylosinbioactive metal complexes against bacterial strains and Candida albicans.

[Zn₂(TYS)₂Cl₂(H₂O)₄]·25H₂O and [Cu₂(TYS)₂Cl₂(H₂O)₄]·25H₂O complexes revealed low concentration value of MIC results with S. aureus with the same value for both 31.25 µg/ml. For B. cereus species both novels tested complexes Zn(II)-TYS and Cu(II)-TYS show lower MIC values with higher antimicrobial activity with value 15.625 µg/ml. Both tested metal complexes of tylosin possess the highest activity with the lowest or equal concentration rather than the tylosin ligand that is sufficient to make the effect of bacterial inhibition.

Free macrolide ligandtylosin show lower MIC value for the fungal strain Candida albicans with value 62.5 µg/ml than two complexes, Zn(II)-TYS and Cu(II)-TYS which poses the highest antifungal activity has the same MIC value by 125 µg/ml. It means that the ligand has better MIC value than Zn(II)-TYS and Cu(II)-TYS complexes although both complexes have higher antifungal activity than tylosin ligand.

5. Conclusion

Elemental analysis and spectroscopic measurements of IR, UV/Vis, and ESR are applied to prove the structures of novel tylosin metal chelates, which are then confirmed by thermal analyses. The analytical results were used to determine the stoichiometry of complexes. Spectroscopic Elucidation of complexes confirms tetragonal distortion geometries. An ESR spectrum of copper was studied for Cu(II)-TYS complex, which is compatible with electronic studies. The spectral data confirmed that tylosin acts as a bidentate ligand. The kinetic and thermodynamic parameters were calculated from the differential thermal analysis curves. Some complexes showed highly antibacterial and antifungal activity against some strains than free ligand. The value of MIC concluded that these metal complexes [Zn(II) and Cu(II)] could be used as effective drugs in killing bacteria and fungi in the field of pharmaceutical applications.

Conflicts of Interest

The authors declare no conflicts of interest regarding the publication of this paper.

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