IHJPAS. 36 (4) 2023 207 This work is licensed under a Creative Commons Attribution 4.0 International License *CorrespondingAuthor: asmaa.idrees1205m@sc.uobaghdad.edu.iq Abstract In this paper, we have provided a very thorough analysis of a new novel chelate metal ion complex of [Cu(II),Ag(I)] prepared via the interaction with the ligand{ 2-amino-8-((4-chloro- 3-hydroxyphenyl) diazenyl)azo]guanine} [LAAG], which is synthesized by diazo coupling of the 5-amino-2-chlorophenol with amino acid guanine. The ligand and its complexes are identified by a variety of techniques, like [HNMR, FTIR, and Uv-vis] spectral, thermal analysis (TGA), and element analyses (CHN). The molar ratio was achieved so that the Cu(II) complex has (1:2) (M:L) with octahedral geometry; however, the Ag(I) complex has (1:1) (M:L) with tetrahedral geometry, and the ligand acts as neutral N,N-bidentate; as well as the ligand (LAAG) and its complexes were assessed against the two types of bacteria (Klebsiella pneumonia, Staphylococcus aureus,antifungal (Candida), and antioxidant This study showed that all compounds (the ligand and its complexes) had antimicrobial activity and more biological activity. Keywords: Azo-dyes, antibacterial, antifungal, Antioxidant. 1. Introduction The synthesis of azo dye derivatives incorporating heterocycles as possible scaffolds is currently receiving a lot of attention in the pharmaceutical industry. A straightforward synthetic method that can produce a variety of azo dye derivatives is required by pharmaceuticals and medical drugs. The target derivatives' bioactive characteristics have been enhanced by the addition of the heterocyclic moiety to the azo dye scaffold. By adding doi.org/10.30526/36.4.3134 Article history: Received 8 December 2022, Accepted 2 January 2023, Published in October 2023. Ibn Al-Haitham Journal for Pure and Applied Sciences Journal homepage: jih.uobaghdad.edu.iq Antioxidant, Antimicrobial and Spectroscopic Discussion of Guanine Azo Ligand with Cu(II) and Ag (I) Complexes Asmaa Edrees Fadhil * Department of Chemistry, College of Sciences, University of Baghdad, Baghdad ,Iraq. Alyaa Khider Abbas Department of Chemistry, College of Sciences, University of Baghdad, Baghdad, Iraq. https://creativecommons.org/licenses/by/4.0/ mailto:asmaa.idrees1205m@sc.uobaghdad.edu.iq mailto:asmaa.idrees1205m@sc.uobaghdad.edu.iq mailto:alyaa.abbas@sc.uobaghdad.edu.iq IHJPAS. 36 (4) 2023 208 heterocyclic moieties, it is simple to adjust the many biological and pharmacological applications of medications, such as their anti-fungal and anti-bacterial characteristics. To this day, attempts are still being undertaken to find better, more effective, and safe synthesis processes for azo dye derivatives. They are the subject of several scientific studies due to their uses as indicators, therapeutic compounds, and textile dyes. Because they possess biological characteristics such as antibacterial, antifungal, anti-HIV, and anticancer, azo dyes are significant in medicinal chemistry [1]. They are, on the other hand, frequently used as biomaterial structural controllers, optical recording devices, photovoltaic devices, molecular switches, photo electronics, and printing systems. They also play a significant role in food and analytical chemistry. Heterocyclic azo dyes and their metal complexes are among these chemicals that can be engaged in biological reactions such as nitrogen-fixing and RNA inhibition. The identification of the ligand and its complexes was done using several physicochemical and spectroscopic methods. The antioxidant, antibacterial, antifungal, A chemical compound known as a color additive is one that combines chemically with another substance to produce a color. In the production of pharmaceuticals, a variety of organics and dyes are used. In the pharmaceutical industry [2], colors are used for financial, psychological, and practical reasons. Drugs can be distinguished by their colors to help patients understand their strengths, lowering the chance of an overdose or underdose. 2. E imentalexper Always the greatest equipment, materials, and solvent agents were employed. Using a Eure EA 3000 Elemental Analyzer, (C.H.N.) determines the elemental analyses and contents of chosen metal ions in the ligand (LAAG) and its complexes. A SHIMADZU, 8400s spectrophotometer was used to register FT-IR spectra in order to determine a sample's pH. CsI was used in the (250- 4000) cm-1 range. The UV-Vis spectra of all the compounds were examined using the (SHIMADZU 1800 - UV-Vis spectrophotometer). The 1H-NMR spectra were measured using a BRUKER AV 400 Avance-III (400 MHz and 100 MHz). The amount of metal in the produced ligand and complexes was quantified using thermal gravimetric analysis (TGA) (SDT, Q600 V20.9 Build). With the aid of Gallenkamp's melting point equipment, the melting points of each chemical were identified. The molar conductance of metal ion complexes was tested in unionized, pure water (10-3 M) (10-3 M)[25,26]. The concentration of chloride in the complexes under examination was determined using the Mohr method. A Sherwood Scientific Auto Magnetic Susceptibility Balance Model was used to test the studied complexes' magnetic susceptibilities at room temperature. IHJPAS. 36 (4) 2023 209 Synthesis of 2-amino-8-((4-chloro-3-hydroxyphenyl)diazenyl)azo]guanine [LAAG] ligand The ligand [LAAG] was produced using a modified version of the process described in the literature [3]. The bulk of azo dyes are typically produced by diazotizing an aromatic primary amine (5-amino-2-chlorophenol) and then coupling with guanine to produce the ligand [LAAG] [4], Scheme 1, demonstrates the azo ligand production process [5]. Scheme 1: Synthesis of the Ligand LAAG Metal complexes synthesis All compounds were synthesized at a mole ratio of [M:L] [1:1], with the exception of (Cu- LAAG), which had a mole ratio of (1:2). A small volume of deionized distilled water was used to dissolve the ligand LAAG (1 mmole; 0.271 gm). The chosen aqueous solution of metal salts [1 mmole of AgNO3 0.1698 gm and 0.5 mmole, 0.8524 gm of CuCl2.6H2O] was refluxed and dissolved in the deionized distilled water, and the ligand solution was slowly added while stirring [6]. IHJPAS. 36 (4) 2023 210 Table 1. some physical and chemical properties for the ligand (LAAG) AND its complexes' 3. Result and Discussion Mole ratio The most typical technique for ascertaining a complex's composition in solution is the mole ratio approach, which was employed in this case. The process is shown in Figure 1 together with the results of the location operations, and the data is shown in Table 2. The findings showed that the synthetic [Ag(LAAG)(H2O)2]NO3.2H2O complex has a [1:1] ratio. while [Cu(LAAG)2Cl2].H2O has a [M: L] mole ratio of [1:2] Figure 1. Plot of absorbance against mole ratio of (LAAG) ligand and its complexes No. Comp. (M.wt) (gm/mol) Color λmax (nm) M:L Ʌm (S.mol- 1. cm2) % Experimental % (Theoretical) C H N M Cl 1 LAAG(C11N7H10O2 Cl) 306.76 brown 339.00 --- --- 48.66 48.02 4.60 3.71 36.99 36.12 ---- ---- 2 [Ag(LAAG)(H2O)2]NO3.2H2O 548.58 yellowish brown 447.00 1:1 55.7 25.32 25.72 3.59 3.50 19.64 19.10 21.02 21.52 ---- 3 [Cu(LAAG)2Cl2].H2O 784.06 green 597.00 1:2 24.6 37.33 37.02 3.24 3.36 27.19 27.48 8.91 8.85 9.95 9.02 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 A B S. MOLE RATIO CM/C HAG-Ag HAG-CU IHJPAS. 36 (4) 2023 211 Table 2. LAAG-Metal ion solution absorbance vs mole ratio Thermogravimetric Analysis (TGA) The projected and actual phase mass losses are listed in Table 3. In our investigation, argon flow was used to measure weight loss [2] and temperatures between (25 and 800 oC). Learning more about stoichiometry, thermal stability, and whether any compounds can be utilized to compute the decomposed species from the thermal graph are the main goals of thermal analysis [7, 8, and 9]. Table 3. the thermal stability of the synthesized compounds M:L Absorbance (λmax nm) LAAG-Ag 447 LAAG-CU 597 1:0.25 0.088 0.134 1:0.50 0.306 0.092 1:0.75 0.363 0.068 1:1 0.369 0.074 1:1.25 0.356 0.077 1:1.50 0.369 0.094 1:1.75 0.358 0.178 1:2 0.364 0.167 1:2.25 0.366 0.177 1:2.50 0.375 0.159 1:2.75 0.369 0.184 1:3 0.365 0.193 1:3.25 0.375 0.205 1:3.50 0.374 0.178 1:3.75 0.389 0.189 1:4 0.395 0.201 Comp. Molecular formula(molec u lar weight) g/mole Step TG. Range of the decompositi on on (oC ) Suggested Assignme nt % Mass loss Calcula te % Foun d % LAAG C11H10N7O2 Cl 1 25-100 H5Cl 12.16 13.13 2 100-310 H5C5 19.53 21.18 3 310-400 C2 8.847 7.82 4 400-800 C4 N2 24.80 24.77 Residu e >800 N2 O2 34.83 33.25 [Ag(LAAG)(H2O)2]NO3.2 H2O AgC11H18N8O9 1 25-90 H2O 3.508 3.28 2 90-249 H2O.C2H10 9.55 9.47 3 249-390 ClC3H4 13.64 13.76 4 390-760 C6N5 25.92 25.88 5 760-800 N1.5 3.50 3.82 Residu e >800 AgN1.5O7 44.0 43.80 1 25-62 2H2O 3.085 4.15 IHJPAS. 36 (4) 2023 212 Figure 2: Thermogram for the (A)LAAG ligand (B)[Ag(LAAG)(H2O)2]NO3.2H2O (C)[Cu(LAAG)2Cl2] FTIR Spectra All of the generated about the nature of the linkage between the metal ion and the ligands, and the accompanying changes : 1- A band at (1573, 1562) cm-1 in the spectra of the ligand (LAAG) associated with (C=N) in the imidazole ring for guanine [8] [10]. The coordination with the metal ion caused this band to change in location and shape. [figure 3] 2- In the spectrum of the ligand (LAAG), the stretching vibration of the bonds (OH), (N-H), and (C=O) were unaffected in the spectra of the complex [Table 4]. Suggesting that no chelating occurred via these bonds [10], however, small alterations in location or form were occasionally attributed to a decrease or increase in resonance as a result of chelating [11]. 3- The distinct feature bands for the azo compounds ʋ (N=N) this band appeared at (1413) cm-1 in ʋ(N=N) in the spectrum of [LAAG], ʋ (C-N=N-C) at (1373,1342) [Cu(LAAG)2 Cl2].H2O CuC22H24N14O6 Cl2 2 62-200 Cl2.10H 10.09 10.33 3 200-400 Cl2.5H 9.39 10.30 4 400-500 C7.5H 10.79 11.35 5 500-625 C12 18.79 19.36 6 625-700 C3N5 13.46 13.51 7 700-800 N8O2 18.65 18.36 Residu e >800 Cu O2 N 15.85 13.97 A B C IHJPAS. 36 (4) 2023 213 cm-1. The posture and intensity of these bands were minified in the complex spectra by chelating [10, 11] 4- A number of new bands were not present in the free ligand spectra when we were seen. However, the most noticeable changes were in the range [622, 405] cm- 1.These bands, which appeared in this region, may be related to ʋ(M-Nazo), ʋ(M- Nimd) ʋ(M-O)H2O and ʋ(M-Cl).This will support our result as regards the chelation sites of the ligands with metal ions, and from the above, we conclude that the two ligands (LAAG) act as neutral N,N-bidentate ligands, forming a penta chelating ring [12]. Table 4. FTIR assignment bands for LAAG and their complexes w: weak Sh :sharp br: broad s: strong t: triple py: pyrimidine m: medium d:double imd: imidazole vw:very week Com. ʋ(O H) ʋ(NH 2) ʋ (C= O) ʋ(C=N)i md. ʋ(N= N) ʋ(- C- N= N-C- ) ʋ( M- N) imd . (M - N) az o ν (M - Cl ) ν (M O) H2O LAAG 347 2 m 3288 3170 d 169 5 167 2 d 1573 1562 d 1413 137 3 134 2d --- --- --- --- [Ag(LAAG)(H2O)2]NO3 .2H2O 343 3 332 8 d 3182 3110 d 169 9 165 4 161 6 t 1544 w 1487 1458 D 136 3 s 609 51 8 w --- 450 vw [Cu(LAAG)2 Cl2].H2O 343 5 335 3 d-br 3193 3112 d 169 5 167 4 d 1612 1564 d 1415 1377 D 131 5 w 605 51 4 w 40 5 v w --- IHJPAS. 36 (4) 2023 214 Figure 3. FTIR spectra for the (A)LAAG ligand (B)[Ag(LAAG)(H2O)2]NO3.2H2O (C)[Cu(LAAG)2Cl2].H2O The 1H-NMR Spectra Figure 4 shows the chemical shift [𝛿] in (ppm) of the ligand (LAAG) and the Table was listed data (5) [13,14]. Table 5. 1HNMR data for the LAAG ligand Figure 4. 1HNMR Spectrum for the LAAG ligand Comp NHpym.,1H OH,1H Ar,4H NH2,2H imidazole LAAG 10.25 9.5 6.62-7.02 6.63 6.00 B A C IHJPAS. 36 (4) 2023 215 Electronic spectrum of the (LAAG) ligand and its complexes and the magnetic proprieties The electronic spectrum of the ligand (LAAG) in water [10-4 M] with the range (280- 1100)nm was given in Figure 5. It had two bands, the first band at(293nm,34129cm-1) which was associated with the intramolecular transition of heterocyclic and aromatic moieties [15][24]. The second band, which was seen at (339 nm, 29498 cm-1) ( n→π*), was attributed to an intramolecular charge transfer that occurred via the carbonyl and azo moieties. Sharp absorption bands in the electronic spectra of the dia magnetic (d10) Ag(I),[25][29] complex were identified as the charge transfer C.T [16]. Figure 5. The electronic Spectra for: (A) LAAG ligand (B) [Ag(LAAG)(H2O)2]NO3.2H2O (C)[Cu(LAAG)2Cl2] complexes The electronic spectrum of the [Cu(LAAG)2Cl2] H2O complex [figure5 ] was υ1 = [2B1g→ 2A1g] (904nm; 11061cm-1) υ2= [2B1g→ 2B2g] (798 nm; 12531 cm-1) υ3= [2B1g→ 2Eg] (597, 16750 cm-1) The CT was hidden with υ3, this is a defining feature of distorted octahedral because of the Jahn-Teller distortion (D4h). The magnetic moment is 1.33 B.M. in energy nm. 300.00 400.00 600.00 800.00 1100.00 A b s. 1.000 0.800 0.600 0.400 0.200 -0.000 A B C IHJPAS. 36 (4) 2023 216 Table 6. spectrum informationof UV-Vis for (LAAG) ligand and their complexes Antimicrobial Activity We used two types of bacteria to study the biological activity of the ligand [LAAG] and their complexes: Staphylococcus aureus (gram-negative) and Klebsiella pneumonia (gram-positive). with [10-3M] being the solution concentration used as a control for bacteria and fungi, respectively. The solvent utilized was water. Amoxicillin and fluconazole were utilized as the reference drugs to evaluate the efficiency of amoxicillin compared to that of synthetic substances. The substances have demonstrated high efficacy in eliminating germs and fungi. They had varied deactivation capacities against the two types of chosen bacteria, as will be stated below[20][21][30]. For Klebsiella pneumonia,Staphylococcus aureus and candida:- [Ag(LAAG)(H2O)2]NO3.2H2O > [Cu(LAAG)2 Cl2].H2O>LAAG Figure 6. The inhibition zones versus bacterial gram-positive (klebsiella pneumonia) and gram- negative (Staphylococcus aureus) for the ligand (LAAG) and theirs complexes Compound ℷ(𝑛m) Wavenumber (Cm) Assignment hybridization Geometry LAAG 293 339 34129 29498 𝜋 → π ∗ 𝜋→π* --- --- [Ag(LAAG)(H2O)2]NO3 .2H2O 467 22371 CT Sp3 Tetrahedral [Cu(LAAG)2 Cl2].H2O 904 798 597 11061 12531 16750 2B1g→ 2A1g B1g →2B2g B1g →2Eg sp3d2 Distorted octahedral IHJPAS. 36 (4) 2023 217 Figures 7. The inhibition zones versus fungal (Candida albicans) for ligand (LAAG) and their complexes Table 7. Inhibition activity rate of LAAG ligand against and their complexes Antioxidant Scavenging Activity In vitro antioxidant and radical scavenging activity of LAAG ligand and [Ag(LAAG)(H2O)2]NO3.2H2O complex were assessed using reductive ability (antioxidant activity) and DPPH radical scavenging activity [18][23] compared to ascorbic acid C)[17] [19][22]. The ligand was a better antioxidant than the complex. Table 8. DPPH radical scavenging activity of, LAAG ligand, [Ag(LAAG)(H2O)2]NO3.2H2O complexes and vitamin C. Concentration (mg/ml) DPPH Radical Scavenging Activity (Mean ± SD; %) LAAG [Ag(LAAG)(H2O)2]NO3.2H2O Vitamin C 12.5 39.12±1.874 26.77±1.892 42.20±1.408 25 43.87±3.216 42.21±1.771 57.91±3.423 50 47.99±1.057 49.54±1.771 65.20±2.567 100 65.97±0.637 49.54 ±1.597 78.67±1.850 200 73.84±2.670 62.08±1.242 85.03±0.598 Compounds Gram(-) Negative Gram(+) Positive Candida Staphylococcus aureus klebsiella pneumonia Amoxicillin 20 10 --- Fluconazole --- --- 20 LAAG 17 10 16 [Ag(LAAG)(H2O)2]NO3.2H2O 27 15 17 [Cu(LAAG)2 Cl2].H2O 17 11 20 IHJPAS. 36 (4) 2023 218 Figure 8. DPPH radical scavenging activity of (1)LAAG and (2)[Ag(LAAG)(H2O)2]NO3.2H2O 4. Conclusion Two new metal complexes with novel azo ligands derived from guanine by the conventional diazo-coupling reaction Their molecular formulae were asserted by different spectroscopic methods such as [FT-IR, UV-Vis, HNMR ] spectra, thermal analyses (TGA), and C.H.N analyses. 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