IHJPAS. 36(1)2023 260 This work is licensed under a Creative Commons Attribution 4.0 International License Preparation, Characterization, Theoretical and Biological Study of new Complexes with mannich base , 2chloro –N-5-(Piperidin -1-ylmethylthio)-1, 3, 4- Thiadiazol-2-yl)acetamide Abstract A new Mannich base ligand was prepared by reacting the 2-chloro.-N-(5-mercapto-1, 3, 4- thiadazol -2-yl) acetamide and Piperidine in the presence (formaldehyde) (L) ligand. A series of ligand complexes were prepared from (L) with the metal ion Co (II), Ni (II), Cu (II), Pd (II), Pt (IV), and Au (III). Various spectroscopic techniques such as C.H.N.S, FTIR, UV-VIS, , 1HNMR, 13CNMR, Magnetic moment, and molar conductivity successfully characterize the obtained compounds. The M: L ratio was determined using the molar ratio method in solution. All prepared compounds' antibacterial and antifungal activity was studied against two types of bacteria and one type of fungi at a rate of 0.02M. The standard ΔH° f and ΔEb of the ligands and all the prepared complexes were calculated using Hyperchem 8.0.7 program, and the study proved that the complexes are more stable than the ligands. In addition, the vibrational frequencies of the ligand were calculated, and the theoretical error rate of the process was calculated. Keywords: Transition Metal Complexes, Mannich Base, 1,3,4-thiadazole Antibacterial and Antifungal. doi.org/10.30526/36.1.2983 Article history: Received 23 Augest 2022, Accepted 25 September 2022, Published in January 2023. Ibn Al-Haitham Journal for Pure and Applied Sciences Journal homepage: jih.uobaghdad.edu.iq Noor Ali Hasan Department of Chemstry , College of Science for Women, University of Baghdad,Baghdad, Iraq. nour.ali1205a@csw.uobaghdad.edu.iq Shaymaa R. Baqer Department of Chemstry , College of Science for Women, University of Baghdad,Baghdad, Iraq. shyma0213@gmail.com https://creativecommons.org/licenses/by/4.0/ mailto:nour.ali1205a@csw.uobaghdad.edu.iq mailto:shyma0213@gmail.com IHJPAS. 36(1)2023 261 1. Introduction The thiosemicarbazide compound with the molecular formula (CH5N3S) and its complexes are of great biological importance due to their wide uses [1]. The compound thiodiazole compound with the molecular formula (C2H2SN2) is a heterogeneous compound because it contains two atoms of Carbone, Two atoms of nitrogen, and one sulfur atom [2]. Two nitrogen atoms and the sulfur atom are electron-donating [3] and are essential ligands in complexes when they bond with metal elements [2]. It has several types, one of which is the compound 1,3,4- thiadiazole, which is applied as anti-tumor medicine. Several derivatives are used as carbonic anhydrase inhibitors and antiparkinsonian agents [3]. Industrial and biological in the medical field, compound 1,3,4 has many applications against bacteria [4], fungi [5], and cancer [6]. The Mannich reaction is of significant importance reactions in chemistry. It is a reaction that includes three or more components of a substance or solvent [7], and it is one of the early examples of a three-component reaction [8]. In the past years, the focus has been on the complexes of Mannich bases that have heterogeneous rings, taking into account the amino-methyl group and studying the relationship of structure, its effect on the biological activity as anticancer agents and toxic to cancer cells [9,10]. There was also a focus on studying their importance as antibacterial and anti- fungals[11], anticonvulsants, anti-inflammatory [9], analgesic, and antioxidant activities [10]. The complexes of Mannich 's bases are essential and have wide applications in the medical and biological fields [11]. They are used as antioxidants [10], anticancer, antibacterial, and anti- fungal [12], and they have proven their effectiveness in all biological and medical fields [13]. 2. Materials and Methods In this paper, high-purity chemicals were used. CHNS elemental data were measured using an Eager300 elemental analyzer. The mineral content was determined using a Shimadzu 670 Flam Atomic Absorber Spectrophotometer. Conductivity data were acquired at 10-3 M in the DMF solution of the complexes using a WTW conductance meter at 25 °C. FT-IR spectra were measured with a Shimadzu and Perkin Elmer FT infrared spectrophotometer using CsI (4000-200) cm-1. Tablet. Absorption in the UV-visible region was recorded in ethanol solution using UV-Vis. Shimadzu Spectrophotometer 1800pcs. The magnetic susceptibility measurement of the complexes was carried out using the Faraday method, and the magnetic correction factor (D) was calculated using the Pascal constants of the atoms that made up the prepared complexes and the Brukar Magent BM6 device. 1H, 13CNMR for compounds were at 25 °C using the Brukar400MHz . The grade of all prepared compounds was measured by Gallen Kamp MF.B-6. Preparation of starting material 2-amino 5-mercapto 1,3,4 thiadiazole (S1) In a round bottom flask, (2gm,0.02mol) of thiosimcarbazide was dissolved in (25ml) ethanol (0.016gm,0.0002mol) of anhydrous sodium carbonate. (4.712 gm, 0.062 mol) of (CS2) was added with stirring and heating at (40C0) for an hour. Then, the mixture was raised for 7 hours after the solvent was evaporated (50ml) in distilled water, and drops of concentrated hydrochloric acid were added. A greenish-yellow was the precipitate. The color of the precipitate (was yellowish white, with a melting point of (229-231) C° and the molecular formula (C2H3N3S2) [14]. The sediment was washed, and the precipitate was filtered with a quantity of distilled water to get rid of the excess acid. It was recrystallized with the solvent ethanol. IHJPAS. 36(1)2023 262 Preparation of 2-chloro-N-(5-mercapto-1,3,4-thiadiazol-2-yl)acetamide (S2) Dissolved (0.1gm, 0.01M) of 2-amino 5-mercapto 1,3,4 thiadiazole (S1) in ethanol was added with stirring (0.1 g, 0.02M) of chloroacetyl chloride) in an ice bath. The mixture was refluxed with heating for 3 hours. The resulting precipitate was washed with distilled water and recrystallized with ethanol. A yellowish-white precipitate was obtained with a molecular formula C4H4N3S2OCl and a melting point of (250-253) C0. Synthesis of 2- Chloro –N-5-(Piperidine1-ylmethylthio)-1,3,4- Thiadiazol-2-yl)acetamide Ligand (Mannich ligand)(L ) Dissolved of (0.145 gm, 0.04M) of 2-chloro-N-(5-mercapto-1,3,4-thiadiazol-2-yl)acetamide in (10ml) of ethanol solvent was added with stirring and cooling (8ml) from formaldehyde and (0.08g, 0.03M) Piperidine. The mixture was refluxed and heated for six hours, leaving the solution to dry. The sediment was recrystallized in ethanol. The precipitate has a brown color, a molecular formula C10H15ClN4OS2, and a melting point of over 300 C 0. H2N-C-NH.NH2 + CS2 Na 2 CO 3 , C 2 H 5 OH HCl S NN S NH2HS NN S NH2HS ClCH2CCl O NN S N H HS C CH2Cl O 2-chloro-N -(5-mercapto-1,3,4-thiadiazol-2-yl)acetamide 2-amino-5-mercapto-1,3,4 thiadiazole NN S N H HS C CH2Cl O N H HCOH N N S S H NCClH2C O H2 C N 2-chloro-N -(5-(pi peridin -1-ylm ethyl th io)-1,3,4-thiadiazol-2-yl)acetamid e Reflux 3hrs° Reflux 6hrs Scheme 1. Synthesis of Mannich Base, 2chloro –N-5-(Piperidin -1-ylmethylthio)-1, 3, 4- Thiadiazol-2-yl)acetamide (L) 2.2. Synthesis of Complexes In the presence of ethyl alcohol solvent, some Mannich base complexes can be prepared. The ratio 1:1 of CoCl2.6H2O, NiCl2.6H2O, CuCl2.2H2O, PdCl2, H2PtCl6.6H2O, and HAuCl4.H2O and ligand. Then the mixture was refluxed for 3 hours. The resulting solids compounds were filtered off, washed with distilled water and ethanol, and dried in a desiccator. Some physical properties can be observed in Table 1. IHJPAS. 36(1)2023 263 Table 1. The physical properties of color and melting point in addition to the values of C.H.N.S and the percentage of all prepared compounds Elemental analysis Calc. (found) Atomic Abs.% Cal.(Found) Yield % m.p / C0 Color Comp. S N H C 20.85 (21.34) 18.25 (19.01) 4.88 (3.98) 39.10 (38.66) ---- 76 Over 300 Brown Mannich (L) 13.28 (14.00) 11.62 (12.78) 4.15 (3.76) 24.90 (23.78) 12.22 80 118-120 Dark green CoL 13.04 (12.87) 11.00 (10.94) 3.87 (4.08) 24.46 (25.11) 11.96 76 182-185 Light green NiL 12.92 (13.67) 10.90 (11.05) 4.23 (5.12) 24.22 (23.20) 12.81 84 167-170 Dark brown CuL 10.51 (11.41) 8.87 (9.11) 2.46 (3.45) 19.72 (18.77) 32.06 67 218-221 Brown PdL 12.74 (11.93) 10.75 (11.44) 3.38 (4.18) 23.89 (24.02) 21.88 74 202-205 Dark brown PtL 10.48 (11.05) 8.84 (9.94) 2.45 (3.04) 19.62 (18.56) 32.27 77 212-215 Green AuL Theortical study In the theoretical study and using the hyperchem 8.0.7 program, the standard heat of formation and binding energy was calculated for all prepared compounds by PM3, ZINDO\I, and AMBER. The HOMO and LUMO were calculated to determine the active sites in the ligand molecule. The vibration frequencies were calculated theoretically for the ligand PM3 method, the practical results were compared with the theoretical results, and the error ratio between them was calculated, which was acceptable. Antibacterial and Antifungal activity 1.40 g of culture medium for bacteria and fungi was dissolved in a liter of distilled water, as the culture medium used for bacteria (Agar Mueller Hinton) and for fungi (Sabroid Dextroes Agar). 2.After dissolving by heating, the culture medium was placed in the sterilizer for 15 minutes, then poured into sterilized plastic dishes and left to solidify. 3.A hole was made using a cork drill with a diameter of 8 mm to add the material that inhibited the growth of bacteria and fungi. 4.The prepared ligands and complexes dissolved in DMSO at a concentration of 0.02M were injected into the pits of the culture medium. 5.The dishes were placed in the incubator at 37 °C for 24 hour for antibacterial activity and 72 hours for antifungal activity, then the inhibition diameters were measured using a ruler in mm for each of the prepared compounds. 3. Results and Discussion All complexes were soluble in organic solvents DMF & DMSO. The ratio of metal-ligand was (1:1) the ligand, and their metal complexes were characterized: (FT-IR), (UV-vis) (1HNMR 1HNMR and 13CNMR, magnetic susceptibility, and conductivity. IHJPAS. 36(1)2023 264 Fourier transforms spectroscopy (FT-IR) of Mannich ligand (L), and metal complexes FT-IR spectra of all prepared compounds were carried out in 4000 to 200 cm-1. The infrared spectra of Mannich ligand L and metal complexes appeared the active site number at 1701, 720,1161, 2943, 2854, and 1620) cm-1which was due to [(υ CO)υ ,CS))υ , C S C) υ CH2N and C=N1,3,4thio.] sequentially[15].When a metal ion was bonded to the ligand through the nitrogen atom of C=N 1,3, 4thio absorption bands shifted towards frequencies within the range (1602-1658) cm -1 in cobalt, nickel, copper, palladium, platin, and gold complexes, respectively. In other absorption bands, the carbonyl group (C=O) has been shifted according to the frequencies (1728, 1728, 1691, 1690, 1686, 1722) cm-1. In all the prepared complexes oxygen of carbonyl, υ C=O took place in coordination. This supported compatibility with the C=O group [16]. We also noticed a shift in C- S and CSC groups for each of the Co(II), Ni(II), Cu(II), Pd(II), Pt(IV), and Au(III) complexes, which indicated the occurrence of coordination through them. According to these results, the coordination made of this ligand was predicted as a tridentate through νC = N, νC=O group, and sulfur [17]. Other bands could be attributed to νM–N, νM–O, νM–S, and νM–Cl for complexes [13]. Other bands can be shown in Table 2. Also, other bands belonging to the νNH group and the νCH2-N group appeared that they did not have a displacement when the coordination occurred, indicating non-coordination through them [16]. Table 2. The main absorption bands of the infrared spectrum of ligand (L), and its metal complexes (cm-1) ν M-Cl νM-S νM-N νM-O νCS νCSC ν C=N 1,3,4thio. νCH2-N ν C=O COMP. ---- ---- ---- ---- 702 1161 1620 2943 2854 1701 L 345 478 520 586 678 1145 1602 2943 Broad 1728 CoL 337 416 513 547 686 1153 1658 2943 1728 NiL 320 470 513 590 729 1175 1639 2942 1691 CuL 345 482 520 540 77 1171 1608 2947 2854 1690 PdL 310 451 516 559 744 1190 1608 2943 2858 1686 PtL 337 470 516 540 786 1188 1639 2947 1722 AuL Uv-vis Spectra, Magnetic susceptibility and molar conductivity The electronic spectra of all prepared compounds are shown in Table (3). The UV-vis spectrum of the free ligand displayed a sharp band at (25773) cm-1 is attributed to n →π* transition. Transitions appeared in the region (33112) cm-1 and 37314 cm-1 is attributed to π → π*transitions [18]. In the UV- visible spectrum of dark green Co (II) complex, two peaks were observed at (10240, 19230 cm-1) dedicated to 4T1 g → 4T2g, 4T1 g → 4 T2 g (p) transitions sequentially and (28659, 41838) cm-1. It was due to the transition IL → Co CT, which indicated the octahedral geometry of Co (II). Magnetic moment. (4.00) B.M showed a higher orbital contribution. Conductivity measurement in DMF (67 μS.cm-1) showed that the complex was ionic. The electronic spectrum of this complex can be seen in Table (3) [19]. In the UV- visible of Ni (II) complex, three peaks were observed at (10582, 15772, 24875 cm-1). They were dedicated to 3A2g → 3T2g, 3A2g → 3T1g (f), 3A2g → 3T1g (p) sequentially and (26109, 40983) cm-1 was due to IL → NiCT which indicated the octahedral geometry of Ni. The magnetic moment, (3.09) B.M showed a higher orbital contribution [20]Conductivity measurement in DMF (21 μS.cm-1) appearing that the complex was nonionic [19]. Light Green complex showed paramagnetic and high spin octahedral. The electronic spectrum of this complex, can be seen in Table (3). IHJPAS. 36(1)2023 265 In the UV- visible region of the Cu(II) complex, one peak was observed at (15197 cm-1), dedicated to 2Eg → 2T2g, and (27629, 29069, 42918) cm -1 was due to IL → CuCT which indicated the octahedral geometry of Cu (II) [20]. Magnetic moment, (1.78) B.M [21]. Conductivity measurement in DMF (61 μS.cm-1) showed that the complex was ionic. Dark brown complex showed paramagnetic and high spin octahedral. The electronic spectrum of this, complex can be seen in Table (3). In the UV-visible region of the Pd (II) complex, one peak was observed at (24038cm-1), dedicated to 1A1g → 1B1g transition, and (31446, 45248) cm -1 was due to IL → PdCT, which indicated square planer geometry of Pd (II) [19]. Magnetic moment (0) B.M. Conductivity measurement in DMF (70 μS.cm-1) showed that the complex was ionic, low spin and square planer geometry [22]. The electronic spectrum of this complex, can be seen in Table (3). In the UV- visible region of the Pt(II) complex two peaks were observed at (10812, 12050 cm-1) was assigned to 1A1g → 3T2g, (23980 cm -1) was assigned to 1A1g → 1T1g, transitions which indicated the octahedral geometry of Pt (IV). Magnetic moment, (0) B.M [23]. Conductivity measurement in DMF (69 μS.cm-1) showed that the complex was ionic [24]. In the UV- visible region of the Au (III) complex, two peaks were observed at (251889, 30030cm- 1) dedicated to 1A1g → 1B1g and 1A1g → 1Eg transitions sequentially and (40983) cm-1 was due to IL → AuCT which indicated the square planner geometry Au (III). The magnetic moment was (0) B.M [25]. The conductivity measurement in DMF was (88 μS.cm-1) [22] .This complex was ionic, low spin and square planer geometry. The electronic spectrum of this complex can be seen in Table 3 [26]. Table 3. The electronic spectra, μeff of complexes, conductivity and suggested geometry for ligand and its metal complexes Suggested geometry Conductivity μS.cm-1 μeeff B.M. Assignments Absorption Cm-1 Comp. ---- ---- ---- n → π* π → π* π → π* 25773 33112 37314 L Octahedral 67 4.00 (3.87) 4T1g → 4T2g 4T1 g → 4T1g(p) IL → CoCT IL → CoCT 10240 19230 28659 41838 CoL Octahedral 21 3.09 (2.82) 3A2g → 3T2g 3A2g → 1T1g(F) 3A2g → 3T1g (p) IL → Ni CT IL → Ni CT 10582 15772 24875 26109 40983 NiL Octahedral 61 1.78 (1.73) 2Eg → 2T2g IL → Cu CT IL → Cu CT IL → Cu CT 15197 27629 29069 42918 CuL Square planer 73 0.00 1A1g → 1B1g IL → Pd CT IL → Pd CT 24038 31446 45248 PdL Octahedral 69 0.00 1A1g → 3T2g 1A1g → 3T1g 1A1g → 1T1g IL → Pt CT IL → Pt CT IL → Pt CT 108551 12050 23980 26881 34602 45662 PtL Square planer 88 0.00 1A1g → 1B1g 1A1g → 1Eg IL → Au CT 25575 27624 AuL IHJPAS. 36(1)2023 266 The 1H-NMR spectra of 2-Chloro –N-5-(Piperidin-1-ylmethylthio)-1,3,4- thiadiazol-2- yl)acetimde The 1H-NMR spectrum of the L can be observed in table 4 in DMSO-d6. The proton nuclear resonance (1H-NMR) spectrum of ligand showed some signals in ppm. Two signals appeared at (1.54, 1.64) ppm and another signals appeared at (2.42,2.43.2.45) ppm, attributed to the proton of Piperidin [11]. The Proton of CH2-N and CH2-Cl groups appared at (4.49) ppm and (3.97) ppm. The signal at (12.39) ppm was attributed to proton of NH group Table 4. The 13CNMR Spectra of 2-Chloro –N-5-(Piperidine1-ylmethylthio)-1,3,4- thiadiazol-2- yl)acetimde The 13CNMR spectrum of ligand showed some signal. The signal at 43.7 can be assigned to carbon of (CH2-Cl) group.The signals at 23.1,24.4 and 54.1 ppm returned to carbon of CH2 of Piperidin and CH2-N of Piperidin [3]. Other signals appeared at 163.2 and 168.4 can be assigned to carbon of (C-S,1,3,4 thio.) group and carbon of carbonyl group, respectively [4]. Another peak can be observed in table 4. Table 4. 1H and 13C nuclear magnetic resonance spectra of Mannich base ligand (L) 1HNMR 13CNMR 1HNMR(DMSO- d6) δ ppm:2.42,2.43,2.45 (d,2H, CH2 of Piperidine) 1.64 and 1.54 (d,2H, CH2 of Piperidine) ,12.39 (S,H,NH) of amide, 3.97,4.49 (S,H,CH2 alphatic of CH2-N and CH2-Cl respectively Mannich (L) : 13CNMR (DMSO-d6) δppm:43.7 (CH2-Cl), 23.1,24.4 (CH2 of Piperidine), 53.1 (CH2-N of Piperidine) ,54.1 (CH2-N) of methylene group, 163.2 (S-C of 1,3,4 thiadiazole group ,168.4 (C=O group). Figure 1. The proposed geometry of the prepared complexes IHJPAS. 36(1)2023 267 Molar ratio Method In the solution state, the ratio metal to ligand can be calculated using molar ratio method at the maximum wavelength, and at concentration 1x10-3of all the prepared complexes. The results proved that the ratio of metal to ligands was (1:1). Figure 2. Molar ratio plot of, L metal complexes 4. Antibacterial and antifungal Activity The biological activity of all prepared compounds was tested by two types of bacteria and one type of fungi at a concentration of 0.02M. The results obtained for the antibacterial and antifungal of the Mannich ligand and its new complexes are presented in Table 5. The diameter of the inhibition zone was measured in (mm) using amoxicillin and metronidazole as standard drugs for bacteria and fungi, respectively. The ligand and complexes generally showed moderate activity, and some were high towards bacteria and fungi tested. The nickel complex showed higher activity against all bacteria and fungi than standard drugs. This would suggest that the chelation could facilitate the ability of a complex to cross-brand into the cell wall of bacteria and fungi and kill them [27]. Table 5. Antibacterial activity of L and its Metal Complexes at 0.02 M Inhibition Zone (mm.) Fungi Gram negative Gram Positive Compounds Candida Klebsiella E.coli Bacillus staph 24 20 16 29 27 CoL 1 27 22 32 27 35 NiL 2 18 14 22 18 21 CuL 3 21 14 14 17 18 PdL 4 15 13 15 15 20 PtL 5 15 15 20 12 20 AuL 6 -ve -ve -ve -ve -ve DMSO 7 25 21 25 25 28 L 8 ---- 11 13 12 12 Amoxicillin 10 13 ---- ---- ---- ---- Metronidazole 11 IHJPAS. 36(1)2023 268 Table 6. The vibrational frequencies of L using HyperChem-8.0.7 program Symb. ν(C=N) ring νC=O νCSC νCH2-N νCS Experimental 1620 1701 1161 2854 2943 702 Theoretical *1623 1768* 1190* 2928 2998* *739 Percentage of error (0.18) (3.78) (2.43) (-1.49) (1.83) (5.00) Figure 3. Vibration spectra of ligand Mannich base (L) ν NH ν C=O ν CH2 Aroma. ν CH2-N ν C=N ν CH2-alip. ν NH ν C=O ν CH2 Aroma. IHJPAS. 36(1)2023 269 Figure 4. HOMO, LUMO and, Electrostatic Potential of ligand L Table 7. Heat of formation, binding energy (in KJ.mol-1) and dipole, moment (in Debye) for ligand, (L) and it's metal complexes, using HyperChem-8.0.7 program PM3 ZINDO/1 AMBER Comp. ΔH°f ΔEb µ ΔH°f ΔEb µ ΔEb= ΔH°f µ L 17.09737 3146.68162- 1.73 -8705.7206 -12205.5026 3.23 ---- ---- CoL 330.3585- 3953.05357- 4.78 -6958.85848 -10581.55348 9.57 ---- ---- NiL 185.61811- -3673.94011 4.32 -6893.90242 -10382.22442 6.30 ---- ---- CuL 175.42755- 3776.42255- 2.22 -6999.43292 -10600.42792 5.17 ---- ---- PdL ---- ---- ---- ---- ---- ---- 88.4691 3.35 PtL ---- ---- ---- ---- ---- ---- 255.7924 4.06 AuL ---- ---- ---- ---- ---- ---- 89.5963 3.22 5. Conclusion Mannich base ligand complexes have been successfully synthesized using the conventional method. The assembly of the six proposed complexes was successfully achieved by the procedures previously described. The results obtained from this investigation were obtained according to the data presented by the physical and chemical analysis. 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