SYNTHESIS, SPECTROSCOPIC CHARACTERIZATIONS AND DFT STUDIES ON THE METAL COMPLEXES OF AZATHIOPRINE IMMUNOSUPPRESSIVE DRUG

A complex of the immunosuppressive drug azathioprine with Cr(II), Mn(II), Fe(II), Zn(II), Cu(II), Ni(II), and Co(II) were synthesized and characterized through spectroscopic and thermal studies. The infrared spectra show the coordination of azathioprine via N(9) to the metal, also, the range around 640–650 cm remains unchanged in the complexes, indicating the possibility that the ether group may not be involved in the binding. Thermogravimetric analysis (TG), thermogravimetric derivational analysis (DTG), and differential thermogravimetric analysis (DTA) have been studied in the temperature range from 0 °C to 1000 °C. The study of pyrolysis showed that all complexes decompose in more than one step and that the final decomposition product is metal oxide. The DFT (density functional theory) with B3LYP/6-31G++ level of theory was used to study the optimized geometry, HOMO→LUMO energy gap, and molecular electrostatic potential map of azathioprine before and after deprotonation.


INTRODUCTION
Medicinal drugs have different functional groups that can bind to metal ions present in the human body [1,2]. Azathioprine, 6-(1-methyl-4-nitro-5-imidazolithio)-9H-purine is a purine homolog and acts as an immunosuppressive drug used in organ transplantation and autoimmune diseases [3][4][5]. It is also used to treat Crohn's disease and rheumatoid arthritis [6]. It stops the enzyme amido-phosphoribosyl transferase and prevents the synthesis of RNA and DNA from cells [7]. The stability of complexes with medicinal drugs plays an interesting role in their biological and chemical activities. Azathioprine was prepared as a metabolically active but disguised prodrug from 6-mercaptopurine in the hope that addition of a sulfur atom substitute of 6-mercaptopurine would affect the distribution and metabolism of the drug, thus improving the targeting of the drug towards tumor cells [8]. It works like slow-release prodrug for 6-mercaptopurine due to its proximity to the ortho-nitro group and susceptible to be attacked by nucleophiles and sulfhydryl groups in biological media.
One putative mechanism of action of slow-acting antirheumatic medicines calls for the contribution of their copper compounds to destroy extracellular superoxide radicals like superoxide dismutase mimetics [9]. Glutathione breaks down azathioprine in the liver to produce 6-mercaptopurine and 1-methyl-4-5 (S-glutathionyl) imidazole [10]. As a thiopurine, azathioprine acts as a purine antagonist, interfering with biochemical processes involving endogenously occurring purines, which are essential components of RNA, DNA, and some coenzymes [11,12].
Several mineral compounds have been synthesized from azathioprine with the aim of developing a compound with enhanced chemotherapeutic activity and/or selectivity relative to the parent drug [13,14]. Determining which chemical agents favor one binding site over another can influence the design and synthesis of slow-release drugs.
Azathioprine contains a few highly versatile binding sites (a thioether bridge, 4 nitrogen atoms of purine ring, and a nitrogen atom plus a nitro group of an imidazole substituent which are likely to be able to coordinate with metals [15]. On neutral azathioprine as an N(9)-H tautomer in the crystal structure of azathioprine [16]. By deprotonation or tautomerization of azathioprine, all four N atoms in the purine ring become potential coordination sites for metal ions [13,15] cleavage chemically to produce 6-mercaptopurine in red blood cells [17] by the enzyme glutathione-Stransferase [18]. More than 80% of azathioprine is converted to 6-mercaptopurine [19,20]. It's also transformed to 8-hydroxy-azathioprine by the enzyme aldehyde oxidase, which is then converted to an inactive metabolite, 6-thiouric acid, by xanthine oxidase [21]. Minerals in chelated forms are better transported into the cell [22].
In the current study, metal complexes of azathioprine were prepared and characterized with chromium, manganese, iron, cobalt, nickel, copper, and zinc. The primary goal of the present research is to study the thermal and spectral behavior of the synthesized complexes. DFT studies with B-3LYP/ 6-31G++ level of theory was employed to generate optimized structure for azathioprine (AZA) before and after deprotonation (AZA -) with minimal energy and to study the preferential binding sites through MEP map.

Materials
The chemicals and reagents used in this article were received from "Merck Chemical Company" and started in preparation without other purification.

Synthesis
A metal divalent chlorides "MCl2" solution of (0.1 mol, MCl2 = MnCl2, FeCl2, CoCl2, NiCl2, CuCl2, ZnCl2, CrCl2) in methanol was treated under stirring with an aqueous solution of (0.1 mol) azathioprine ( Figure 1). The reaction mixtures were stirred under reflux at 60 o C for 3-4 h, the precipitates were filtered off. The resulting solid product was washed twice with acetone and dried in a vacuum. Yield of corresponding azathioprine metal complexes were 70-80%. The metal salts were used directly without any further purification.

Density functional theory
Gaussian 09RevD.01 [23] was used for DFT (density functional theory) studies. Pople's basic set B3LYP/6-31G++ was applied with gradient corrected correlation [24]. The same basic set was used to obtain the optimized structures. Electrostatic potential map (MEP), LUMO (lowest unoccupied molecular orbital), and HOMO (highest occupied molecular orbital) were obtained [25]. The Frontier Molecular orbitals help in determining the chemical stability of the system. For visualization ChemCraft 1.5 software [26] was used.

RESULTS AND DISCUSSION
All the Cr(II), Mn(II), Fe(II), Co(II), Ni(II), Cu(II), and Zn(II) azathioprine complexes are stable at room temperature, hydrated, insoluble in water and soluble in common organic solvents such as DMF and DMSO. The analytical and physical data of the synthesized complexes are given in Table 1, spectral and thermal stability data (Tables 2 and 3) are compatible with the suggested molecular formula [C9H7N7O2SCl2M].3H2O (M = Cr 2+ , Mn 2+ , Fe 2+ , Co 2+ , Ni 2+ , Cu 2+ , and Zn 2+ ). The molar conductances are in the 10-15 ohm -1 cm 2 mol -1 range, indicating a non-electrolytic nature [2]. Many attempts were made to grow a single crystal but unfortunately, they were failed. Reaction of azathioprine with metal salts (MnCl2, FeCl2, CoCl2, NiCl2, CuCl2, ZnCl2, CrCl2) using (1:1) molar ratios in methanol/H2O (50:50 v/v) gives complexes (1-7). The composition of the complexes formed depends on metal salts, and the molar ratio. The elemental analyses of the complexes listed in the experimental section establish the 1:1 ratio of metal: azathioprine for all the metals used.

IR spectra
The IR spectrum of azathioprine-chromium complex given in Figure 2 (representative spectrum) and tentative band assignments of all complexes are given in Table 2. In the IR spectra of all complexes, no significant peaks were observed in 2500-4000 cm -1 . This indicates deprotonation of N(9) nitrogen upon coordination with metal [27]. After complex formation, a change in band frequencies in the range 1570-1590 cm -1 is observed due to redistribution of electron density of the ring [28,29]. The splitting of band of free ligand at 1234 cm -1 on complexation is due to the coordination of N(9) nitrogen with metal [27]. The band around 640-650 cm -1 remains unshifted in complexes, indicates possibility of non-participation of thioether group from binding [30,31].

ESR spectra
The ESR spectrum of Cu(II)-azathioprine complex was scanned at room temperature. Copperazathioprine complexes displayed an "isotropic" sharp signal (g = 2.097) which is due to less "dipolar interaction" and "spin-lattice relaxation" [32][33][34][35] of the "coordinated ligand" and "spin exchange interaction" between Cu(II) ions [36]. The hyperfine coupling cannot be resolved due to the metal ion (A Cu ). Planar, sp 2 -type conformation, nitrogen shows a large superfine coupling constant (shf) than tetrahedral nitrogen, sp 3 (lower s). The distortion effect from the planar matrix to the tetrahedral matrix in the first coordination domain may also lead to some decrease in the nitrogen shf coupling [37], since it leads to reduced interference between the non-paired copper electron orbital with the orbital and the ligand orbital and thus causes a lower density Rotation on nitrogen and consequently, the nitrogen coupling constants decrease.

H NMR spectra
At room temperature, the 1 H-NMR spectrum of azathioprine [38] in DMSO-d6 shows three peaks at 8.58, 8.55 and 8.25 ppm are assigned to H(2), H (8), and H(11) aromatic protons, respectively. The N(9)-H resonates at 13.88 ppm. The H(11) proton of the 1-methyl-4-nitroimidazole ring is resonated at the upfield of the purine protons. The pyrimidine ring is a π-electron deficient system, while the imidazole ring is a π-electron rich system [39]. The zinc-azathioprine complex shows downfield shift for H(2) and H(8) only while H(11) is observed to be unshifted in comparison to azathioprine [40]. The downfield shifts of the azathioprine protons upon zinc(II) coordination with ring nitrogens of purines, results in shift of electron density, which makes the proton more acidic adjacent to the binding site [40][41][42]. At the same time, due to deprotonation of azathioprine anion (AZA -), localization of electron density at the same binding position makes H(8) shielded [43,44]. Table 2. IR spectral band assignments of azathioprine and its metal complexes.

Thermal studies
To establish a different decomposition process and to confirm the proposed chemical measurement, the thermal behavior of the complexes was investigated. Table 3 summarizes the findings of the thermal analysis. A very good relationship between the calculated and obtained weight loss values were observed. The different species lost in different stages of decomposition are given in Table 3. TG, DTA, and DTG data are in well agreement. All complexes decompose in more than one step giving metal oxide as a final residual product. The TG, DTG, and DTA curves of azathioprine-chromium complex given in figure 3 (representative figure) and tentative band assignments of all complexes are given in Table 3 [45,46]. The order of decomposition reaction in each case is unity.

Magnetic measurements
Gouy's method was utilized for magnetic susceptibility measurements using the following equation 1 [47]: where "Xg= mass susceptibility per gram of sample", "C is the calibration constant of the instrument", "R is the balance reading for the sample and tube", "R0 is the balance reading for the empty tube", "M is the weight of the sample in grams" and "T is the absolute temperature". Magnetic measurements were performed on a Sherwood scientific magnetic balance according to the Gouy's method [47]. , respectively. This may be due to Jahn Teller distortion, which results in distortion in structure from octahedral geometry [48].

DFT studies
The optimized structures of azathioprine were obtained through B3LYP/6-31G++ level of theory. Azathioprine has -1278.611130 a.u. minimum SCF energy after 23 steps of optimization, while after deprotonation it was found to be -1277.895903 a.u. after 23 optimization steps. The optimized structure with strain-free lattice constants of azathioprine (AZA and AZA -) is shown in Figure 4. The electrostatic potentials strength of azathioprine before and after deprotonation is represented by MEP map (molecular electrostatic potential map) ( Figure 5). Electropositive region is displayed with blue color and electronegative with red. The most electropositive region is around O2 and O3 atoms, while the most electronegative region is around N8 atom of azathioprine ( Figure 5a). These results signify the higher tendency of deprotonation of N8 atom and be available as a preferential binding site for the electrophilic and nucleophilic attacks [49]. Figure 5b shows the creation of electronegative region around N8 atom due to deprotonation. The MEP surface maps are plotted from deep red as -7.928 e -2 to deep blue as +7.928 e -2 and -7.928 e -2 to +7.928 e -2 in the color scale for azathioprine before and after protonation of N8 atom, respectively [50]. Frontier molecular orbitals (FMOs) were obtained through DFT calculations. Electron donors are highest occupied molecular orbitals (HOMO), while electron acceptors are lowest unoccupied molecular orbitals (LUMO). According to the literature the compounds having smaller energy gap are soft in nature, possess low kinetic stability, and higher chemical reactivity [50]. Figure 6 represents the energies associated with LUMO and HOMO with HOMO-LUMO gap. As it can be seen in Table 4, The HOMO-LUMO gap (∆E) for azathioprine is 4.3373 eV and for azathioprine after deprotonation of N8 atom, ∆E = 4.3541 eV [50], total dipole moment of the molecules (μ) and polarizability (α) were also obtained.  Figure 4. Optimized structure with Mulliken atom numbering of (a) Azathioprine and (b) azathioprine after deprotonation of N8 atom.