Synthesis of three new macroacyclic Schiff base complexes and the investigation of their anticancer, antioxidant and antibacterial properties: theoretical and solid state structures

doi:10.21203/rs.3.rs-2061704/v1

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Synthesis of three new macroacyclic Schiff base complexes and the investigation of their anticancer, antioxidant and antibacterial properties: theoretical and solid state structures

https://doi.org/10.21203/rs.3.rs-2061704/v1

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Three symmetrical macroacyclic Schiff base complexes were prepared from a ligand derived from the condensation reaction of 1,4-bis(2-Aminophenyl)piperazine and salicylaldehyde (L) with Cu(II), Ni(II) and Co(II) perchlorates. The ligand and the Schiff base complexes were characterized by elemental analyses, FT-IR and mass spectrometry, with the structure of [CoL] being determined by a single crystal X-ray structural analysis. In this complex, the cobalt is in a distorted trigonal prismatic coordination environment, surrounded by the six donor atoms of the deprotonated hexadentate ligand. In order to compare the experimental and theoretical data and determine structure parameters in all complexes, Density Functional Theory (DFT) calculations at the B3lyp and BP86 levels with Def2-TZVP basis set have been carried out. NBO and QTAIM analyses have been used to describe the nature of M-O and M-N bonding in these complexes. The synthesized complexes were screened for their antioxidant activities using the DPPH free radical scavenging assay while their bactericidal activity was evaluated by the paper disc diffusion method against both Gram-negative and Gram-positive bacteria, revealing strong antioxidant activities and moderate effectiveness against all bacteria tested. The cytotoxicity of the metal complexes was also investigated against AGS (gastric) and A549 (lung) adenocarcinoma cells.

Biological sciences/Biochemistry

Biological sciences/Computational biology and bioinformatics

Macroacyclic

X-ray structural

DFT calculations

Anticancer

Antioxidant

Antibacterial

Schiff-base complexes can be considered as the one of the most significant stereochemical models in transition metal coordination chemistry due to the fact that they are structurally diverse and are easily prepared [1, 2]. They have a very significant role in the development of coordination chemistry which has resulted in a wide variety of research ranging from physicochemical and biochemical studies to purely synthetic studies [3]. There has been much research into metal complexes with chelate ligands with the aim of mimicking the reactions found in biochemical systems, including the redox functions of different metalloenzymes and the coordination and reactivity of dioxygen. Metalloenzymes can have specific functions, such as redox [4–9] and structural and catalytic roles [10–14] and accordingly there has been intensive research into metal complexes that can mimick the active sites of metalloproteins. In order for the metal to have a similar coordination environment to those found in proteins, many model metal complexes have obtained using sterically bulky polydentate ligands. 2-Hydroxy Schiff base ligands and their complexes derived from the reaction of derivatives of salicylaldehyde with amines have been extensively studied in great details for their various crystallographic, structural and magnetic features [15–16]. Schiff bases and their metal complexes have been studied for their important bio-medicinal properties including antibacterial, fungicidal, antioxidant, anticancer, and herbicidal activties [17–18]. The origin of some pathophysiological abnormalities in the human body, including cancers, is free radicals (∗OH⁻ and ∗O₂⁻). Antioxidants were used to treat the human body from such abnormalities [19–20]. Thus it is important to make the potential drugs with both strong antioxidant and anticancer properties for quick remedy from the miscellaneous type of cancers. In recent years our research group has reported on the synthesis and biological properties of a number of macrocyclic and macro-acyclic Schiff base ligands incorporating the piperazine group together with their metal complexes [20–25]. Of particular interest has been those hexadentate ligands which have a potentially N₄O₂ donor set [26]. In the current study, we have extended this work by the synthesis of a new hexadentate Schiff base ligand from the condensation of 1,4-bis(2-Aminophenyl)piperazine with Salicylaldehyde and prepared the Co(II), Ni(II) and Cu(II) complexes. The cytotoxicity of the complexes were then investigated against AGS (gastric) and A549 (lung) adenocarcinoma cells, their antioxidant activity was studied by the in vitro DPPH free radical scavenging method while their antibacterial activity was measured against a variety of bacterial strains. To enable a comparison between experimental and theoretical data and determine structure parameters Density functional theory (DFT) calculations been utilized while NBO and QTAIM analyses have been used to describe the nature of M-O and M-N (M = Co, Ni and Cu) bonding in these complexes.

2.1. Materials

Salicylaldehyde, 1-Fluoro-2-nitrobenzene, piperazine, metal salts and solvents were commercial products from Merck and used without further purification.

Caution! Perchlorate salts are potentially explosive. Only small amounts of materials should be prepared, with caution, and handled in small quantities.

2.2. Physical measurements

Infrared spectra were collected using KBr pellets on a BIO-RAD FTS-40A spectrophotometer (4000–400 cm^− 1). CHN analyses were carried out using a Perkin-Elmer, CHNS/O elemental analyzer model 2400 series 2. ¹H and ¹³C NMR spectra were taken in CDCl₃ on a Bruker Avance 400 MHz spectrometer using Si(CH₃)₄ as an internal standard. Electron impact mass spectra were performed on an Agilent technologies (HP) 5973 mass (EI at 70 eV) spectrometer.

2.3. X-ray crystal structure determination

Orange single crystals of formula [C₃₀H₂₆N₄O₂Co] were crystallized by vapour diffusion of ether into methanol. Data were collected at 100 K at the MX2 beamline at the Australian Synchrotron, fitted with a silicon double crystal monochromator and Dectris Eiger 16M detector, the wavelength being tuned to approximate Mo-Kα radiation (λ = 0.710872 Å) [27]. Data reduction was performed with XDS [28] using multi-scan absorption corrections. Using Olex2 [29], the structure was solved with the ShelXT [30] structure solution program using Intrinsic Phasing and refined with the ShelXL [31] refinement package using Least Squares minimization on F², using all data. All non-hydrogen atoms were refined with anisotropic displacement parameters, while all hydrogen atoms were placed at geometrical estimates and refined using the riding model. The maximum and minimum electron density peaks were 0.43 and − 0.59 e Å⁻³. Crystallographic Data are given in Table 1. Selected bond lengths and angles are given in Table 2.

2.4. Computational methods

The geometries of all complexes studied here, were fully optimized in the gas phase at the B3lyp [32] and BP86 [33–34] levels of theory with Def2-TZVP basis set [35] using Gaussian 09 package [36]. Vibrational frequencies analysis was computed at the same levels of theory show that optimized structures of all complexes are at the stationary points without any imaginary frequency. The crystal structure coordinates of the Co(II) complex [CoL] was used as the basis for the DFT calculations for the three [ML] (M = Co, Ni and Cu) complexes. It should be noted that the Co(II) complex in high-spin state and Ni(II) complex in low-spin state were studied using density functional methods. The nature of coordinated bonds has been investigated using Atoms-In-Molecules (AIM) [37] at the BP86/TZVP level of theory. The topological parameters of the QTAIM analysis were calculated using the AIM2000 program [38]. Also, Natural Bond Orbital (NBO) analysis was carried out with the internal module Gaussian 09 at the BP86/TZVP level of theory [39].

2.5. Cytotoxicity in vitro

Gastric Cell Line (AGS; IBRC C10071) and lung cell line (A549; IBRC C10080) were purchased from the Iranian Biological Resource Center (IBRC; Tehran, Iran). DMEM: Ham’s F12, used in culturing the lung cancer cell line and Gastric cell line, was supplemented with L-glutamine, 10% Fetal Bovine Serum (FBS), 100 units mL^− 1 penicillin and 100 µg mL^− 1 streptomycin. The cells were kept in a humidified atmosphere at 37°C with a concentration of 5% CO₂. Standard solutions were made by dissolving the compounds in DMSO to give a concentration of 20 mM L^− 1. These solutions were then diluted to give concentrations of 1.56, 3.13, 6.25, 12.50, 25 and 50 mM L^− 1 which were subsequently added to the culture medium. Assessment of cell viability was performed using the modified cell viability (MTT) method in which the reduction of MTT (3-[4, 5-dimethylthiazolyl]-2,5-diphenyltetrazolium bromide) was detected. In each well of a 96-well plate 25 was allocated 5 × 10⁴ cells with 100 µL RPMI medium supplemented with 10% FBS. After allowing 24 hours for cell adhesion, diluted solutions of the tested compounds (0 to 50 µM) were added. In order to prevent the toxicity of the solvent, 0.5% (v/v) was chosen as the final DMSO concentration. After a further 12 h. 10 µL MTT was added followed by incubating the plates at 37°C for an additional 4 h. The formazan blue that was subsequently formed was dissolved in DMSO (100 µL) and the optical density (OD) of the solution in each well was measured at 490 nm using an ELISA plate reader to determine the cell survival curves. Each test was carried out in triplicate and the mean value was used to determine the IC₅₀ value – the concentration of the tested compound which inhibits the growth of the cells by 50%.

2.6. Antioxidant property

The electron donation abilities of the complexes were evaluated by bleaching of the purple-colored chloroform solution of DPPH (2,2-diphenyl-1-picrylhydrazyl) [9]. Briefly, 2.5 mL of different concentrations of each sample (0.2–1 mg mL^− 1 in methanol) were added to 1 mL of 0.3 mM DPPH solution and the mixture incubated for 30 min at room temperature in the dark. The reduction of free radicals was measured by reading the absorbance at 517 nm. Ascorbic acid and quercetin, standard antioxidants, were used as standards. The percentage inhibition of the production of free radicals by DPPH (I %) was calculated using the following formula:

I% = [1– (As – Ab)/Ac] × 100

Where, As is the absorbance of the reaction mixture containing 2.5 mL of sample + 1 mL of DPPH, Ab is the absorbance of the reaction mixture containing 2.5 mL of sample + 1 mL methanol, and Ac is the absorbance of the control sample containing 1 mL of DPPH + 2.5 ml methanol. The IC₅₀ value, defined as the concentration of the sample leading to 50% reduction of the initial DPPH concentration, was calculated from the linear regression plot of the concentration of the test sample against the mean percentage of the antioxidant activity [40].

Table 1

Crystal data and structure refinement for the complex [CoL]
CCDC number	2109477
Empirical formula	C₃₀H₂₆CoN₄O₂
Formula weight	533.48
Temperature/K	100.0(2)
Crystal system	monoclinic
Space group	C2/c
a/Å	19.669(4)
b/Å	7.3570(15)
c/Å	17.509(4)
α/°	90
β/°	104.87(3)
γ/°	90
Volume/Å3	2448.8(9)
Z	4
ρcalcg/cm3	1.447
µ/mm 1	0.737
F(000)	1108.0
Crystal size/mm3	0.1 × 0.05 × 0.015
Radiation	Synchrotron (λ = 0.71087)
2Θ range for data collection/°	5.564 to 64.148
Index ranges	-27 ≤ h ≤ 27, -10 ≤ k ≤ 10, -26 ≤ l ≤ 25
Reflections collected	43102
Independent reflections	3866 [Rint = 0.0358, Rsigma = 0.0164]
Data/restraints/parameters	3866/0/168
Goodness-of-fit on F2	1.156
Final R indexes [I > = 2σ (I)]	R1 = 0.0315, wR2 = 0.0961
Final R indexes [all data]	R1 = 0.0321, wR2 = 0.0966
Largest diff. peak/hole / e Å-3	0.43/-0.59

Table 2

Bond lengths (Å) and Selected Angles (°) for [CoL]
Atom- AtomLength/Å
Co1-O11.9769(9) Co1-N12.1228(11) Co1N22.2874(11)
Atom-Atom-Atom Angle/˚
O1-Co1-O1ⁱ 127.42(6) O1-Co1-N186.82(5) O1-Co1-N1ⁱ87.74(4) O1ⁱ-Co1-N293.69(4) O1-Co1-N2133.26(4) N1ⁱ-Co1-N1167.70(6) N1-Co1-N272.03(4) N1-Co1-N2ⁱ119.33(4) N2-Co1-N2ⁱ64.35(5)
symmetry code i: -x, y, ½-z.

2.7. Antibacterial activity

The antibacterial activity of synthesized compounds was studied against two Gram-positive (Staphylococcus aureus, and Bacillus subtilis) and two Gram-negative bacteria (Escherichia coli, and klebsiella oxytoca). Each of the complexes was dissolved in methanol to give a concentration of 1 mg.ml^− 1 which was then filtered through a 0.45 µm Millipore. Each solution was then added to a suspension of 1.5×10⁸ bacteria.ml^− 1 (10 ml) which were then spread out on the agar medium. Vancomycin, Tetracycline, Ampicillin, and Penicillin were also applied as antibiotic standards. The volume of swelling from the edge and the diameter of the inhibition region of each sample was given in mm.

2.8. Synthesis

Ligand synthesis (H ₂ L)

1,4-bis(2-Aminophenyl)piperazine (A) was synthesized according to the literature procedure [41]. A solution of Salicylaldehyde (1 mmol, 0.15 g) in absolute ethanol (30 mL) was added drop wise to a solution of A (0.5 mmol, 0.13 g) in absolute ethanol (20 mL) and the mixture was gently refluxed and stirred overnight. The resulting product was then filtered off, washed with ethanol and dried in vacuum (Scheme 1).

Yield: 0.19 g (84%). Anal. Calc. for C₃₀H₂₈N₄O₂ (MW: 476.57): C, 75.61; H, 5.92; N, 11.76 Found: C, 75.52; H, 6.08; N, 11.64. EI-MS ( m/z ): 476.4 IR (KBr, cm ^− 1): 3429 ѵ (OH), 1618 ѵ ( C = N ). ¹H NMR (CDCl₃–d6, ppm) δH = 2.55–2.86 (s, H ), 6.58–7.88(H_aromatic), 8.62 (H_imini), 13.43 (OH). ¹C NMR(CDCl₃, ppm): δ c: 55.8–57.8(C_py), 115.8–149.8(C_aromatic), 160.6(C_imini), 161.4(C-OH).

Synthesis of complexes.

General procedure: in order to prepare the metal complexes, the ligand (L) (1 mmol), dissolved in 25 ml of methanol, was added to a solution of M(ClO₄)₂.6H₂O (1 mmol) (M: Ni, Co, Cu) dissolved in 20 ml of methanol. The solution was then refluxed for 24 h, after which triethylamine (3 drops) was added and the reaction mixture was allowed to cool to room temperature. The precipitate was filtered off, washed with excess of ethanol and then dried in vacuum. The products were characterized as the pure compounds (Scheme 2).

[CoL]: Orange single crystals. Yield: 0.45 g (76%). Anal. Calc. for C₃₀H₂₆CoN₄O₂ (MW: 533.49): C, 67.54; H, 4.91; N, 10.50. Found: C, 67.47; H, 4.75; N, 10.32. EI-MS ( m/z ): 533IR (KBr, cm^− 1): 1611 ѵ ( C = N ).

[NiL]: green powder. Yield: 0.5 g (82.3%). M.p. 315°C. Anal. Calc. for C₃₀H₂₆NiN₄O₂ (MW: 533.25): C, 67.57; H, 4.91; N, 10.51. Found: C, 67.49; H, 4.83; 10.42%. IR (cm^− 1, KBr): 1610 (s, m C = N). MS (EI): (m/z) = 533.19

[CuL]: green powder. Yield: 0.49 g (87%). Anal. Calc. for C₃₀H₂₆CuN₄O₂ (MW: 537.14): C, 66.96; H, 4.87; N, 10.41. Found: C, 66.79; H, 4.71; N, 10.32. EI-MS ( m/z ): 537.09 IR (KBr, cm^− 1): 1607 ѵ ( C = N )

We prepared the hexadentate Schiff base ligand with a N₄O₂ donor set by condensation of 1,4-bis(2-Aminophenyl)piperazine, with 2-hydroxy-banzaldehyde in a 1:2 ratio. Elemental analysis, IR, mass, ¹H and ¹³C NMR spectroscopy were used to characterize the formation of the ligand (L). The infrared spectrum shows a strong absorption band at ~ 1618cm^− 1, assigned to the C = N stretching vibration, and with the absence of bands due to C = O and NH₂ stretching vibrations, indicates the formation of the Schiff-base ligand. The mass spectrum of the ligand shows the expected peak at 476.4 m/z. The Ni(II), Co(II) and Cu(II) complexes were prepared by direct reaction of the ligand and metal salts in a 1:1 ratio in methanol and characterized by Mass and IR spectroscopy and by elemental analysis. In the IR spectra the imine stretching bands found for the Ni(II), Co(II) and Cu(II) complexes, 1610 cm^− 1, 1611 cm^− 1 and 1607 cm^− 1, respectively, were shifted to lower frequency compared to the free ligand (1618 cm^− 1) consistent with the coordination of the imine nitrogen to the metal ions. Furthermore, there no absorption bands in the 3400–3300 cm^− 1 region, due O-H stretching bands, confirming that the deprotonation of the phenol had occured .The ligand and complexes are soluble in most organic solvents.

3.1. X-ray structure

The structure of [CoL] is almost identical to that found for [2,2'-{(piperazine-1,4-diyl)bis[(2,1-phenylene)(azanylylidene)methanylylidene]}bis(6-methoxyphenolato)]-cobalt(ii) [CoL¹] [44], a cobalt complex of the hexadentate ligand that has contains methoxy groups attached to the phenol rings. In the present compound the cobalt atom lies on a 2-fold axis, so that the asymmetric unit comprises half the molecule. As with [CoL¹], the cobalt atom is in a distorted trigonal prismatic coordination environment (Figs. 1 and 2), surrounded by the six donor atoms of the deprotonated hexadentate ligand. The absence of a counter ion and the values of the Co-O and Co-N bond lengths are consistent with the oxidation state being Co(II). The atoms N1, N2 and O1ⁱ and O1, N1ⁱ and N2ⁱ (symmetry code i: -x, y, ½-z) form the two triangular faces, which are almost coplanar, the dihedral angle being 6.81(8)°, while the angles between the rectangular faces are 56.21(4)°, 61.90(3)° and 61.90(3)°. Continuous Shape Measure (CShM) calculations, based on a trigonal prismatic geometry [45], give a value of 2.536 (compared to 2.099 for [CoL¹]). The Co-O distances are 1.9769(9) Å, while the Co-N_piperazine distances, 2.2874(11) Å, are longer than the Co-N_imine distances of 2.1228(11) Å, similar to that observed for [CoL¹]. Trigonal prismatic coordination for metal complexes is rare [46], a search of the Cambridge Structural Database (CSD Version 5.43 June 2022 Update) [47] found only 11 cobalt(II) structures with a N₂O₄ trigonal prismatic coordination environment (out of a total of 151 trigonal prismatic Co complexes), only one of which, ((2,2'-((piperazine-1,4-diyl)bis(propane-3,1-diyl(nitrilo)methylylidene))bis(6-methoxyphenolato))-cobalt(ii)) [48], had a N₃O donor set in each of the rectangular faces, as found here for the present complex. The cobalt atom lies in the plane of the atoms of the two (aminomethyl)phenol groups, while the 5-membered metallocyclic rings are in an envelope conformation with the Co atom lying out of the plane of the other 4 atoms. The dihedral angle between the two (aminomethyl)phenol groups is 59.71(3)° while the dihedral angle between the diaminophenyl rings is 72.34(5)°. There are no hydrogen bonds in the structure.

3.2. Theoretical studies

The optimized structures of the ML (M = Co, Ni and Cu) complexes, at the B3lyp and BP86 levels with the Def2-TZVP basis set, are shown in Fig. 3; the starting point for the calculations were the crystal structure coordinates of the Co(II) complex. The calculations show that the optimized structure of the CoL complex is similar to its X-ray structure, in which the two oxygen atoms and four nitrogen atoms of the macroacyclic ligand are coordinated to the Co atom in a distorted Trigonal-Prismatic environment. Selected calculated bond lengths and bond angles of optimized structures of ML (M = Co, Ni and Cu) complexes and corresponding experimental values obtained from the X-ray crystal are reported in Table 3. A comparison between experimental and calculated bond lengths values for CoL complex show that there is a good agreement between the experimental and calculated values using both B3lyp and BP86, but that BP86 shows slightly better agreement than B3lyp (Table 3). For example, as can be seen in Table 1, the calculated bond length values using BP86 for the Co←O and Co←N2 bonds are about 0.031Ǻ and 0.065Ǻ greater than experimental values, respectively, while the Co←N1 bond length is similar to the experimental value. For B3lyp the values for the Co←O, Co←N2 and Co←N1 bond lengths are about 0.031Ǻ, 0.096Ǻ and 0.039Ǻ greater than experimental values, respectively. A comparison of the experimental and calculated bond angles for CoL show small differences. For example, the calculated values of the bond angles differ from the corresponding experimental values by around 0.09º-3.5º for BP86 and around 0.48º-7.7º for B3lyp. A can also be seen in Table 3, changing the M atom from Co(II) to Ni(II) and Cu(II), leads to a decrease in the values of the M←O and M←N1 bond lengths and an increase in the values of the M←N2 bond length. The QTAIM and NBO analyses were also used to characterize the type and nature of M←O and M←N bonds in the ML (M = Co, Ni and Cu) complexes. The results of AIM and NBO analyses were summarized in Table 4. The NBO results for all complexes studied here, demonstrate that the M (M = Co, Ni and Cu) atom has a positive charge which is an acceptor atom and two oxygen atoms and four nitrogen atoms of the macroacyclic ligand as donor atoms have negative charge. As can be seen, the amount of the charge transfer from L ligand to M metal ions are about 1.12e, 1.40e and 1.11e in the CoL, NiL and CuL complexes, respectively. These results also show that in the above complexes, maximum charge transfer occurs between M and L atoms in M←O and M←N1 bonds and minimum charge transfer occurs in M←N2 bond. The Wiberg bond indices (WBIs) for are also reported in Table 4. The largest values of WBI are attributed to M←O and M←N1 bonds and the smallest ones are attributed to M←N2 bonds. Also, the AIM analysis was used to investigate the bond critical points (BCPs) at M←O and M←N bonds bond for above complexes. The topological properties calculated at bond critical points for M←O and M←N bonds are listed in Table 4. The AIM results show that there is a good agreement between the amounts of electron density \({\rho }\left({\text{r}}_{\text{c}}\right)\) of M←O and M←N bonds with calculated WBI in above complexes. Also, the amounts of\({\nabla }^{2}{\rho }\left({\text{r}}_{\text{c}}\right)\) and -Gc/Vc for the M←O and M←N bonds show that the nature of these bonds is partly covalent (of\({\nabla }^{2}{\rho }\left({\text{r}}_{\text{c}}\right)>o\) and -Gc/Vc < 1).

Table 3

The experimental and calculated important Bond lengths (Å) and Bond angles (°) of ML (M = Co, Ni and Cu) complexes.
M-L Bond	CoL			NiL		CuL
M-L Bond	Bond lengths			Bond lengths		Bond lengths
	Experimental	B3LYP/TZVP	BP86/TZVP	B3LYP/TZVP	BP86/TZVP	B3LYP/TZVP		BP86/TZVP
M-O1	1.9769	2.00763	2.00825	1.87339	1.87510	1.94832		1.96688
M-N1	2.1228	2.16204	2.11821	1.94185	1.91793	2.03740		2.01885
M-N2	2.2874	2.38339	2.35270	2.97098	2.98149	2.958		2.89248
	Bond angles			Bond angles			Bond angles
O1-M1-O1ⁱ	127.42	135.147	130.977	168.430	168.009		152.680	151.724
O1-M1-N1	86.82	85.549	86.915	91.586	92.398		90.435	91.010
O1-M1-N1ⁱ	87.74	90.091	87.946	89.067	88.273		91.468	90.969
O1ⁱ-M1-N2	93.69	92.706	92.888	81.305	80.885		88.123	88.143
O1-M1-N2	133.26	127.102	130.894	109.444	110.287		117.172	118.100
N1ⁱ-M1-N1	167.70	168.564	167.594	173.510	173.582		171.939	171.887
N1-M1-N2	72.03	70.901	71.816	65.534	65.822		65.047	65.266
N1-M1-N2ⁱ	119.33	119.806	119.715	108.185	107.978		107.197	106.940
N2-M1-N2ⁱ	64.35	63.034	63.644	51.338	51.314		52.331	52.248

Table 4

Natural charges of M, N and O atoms, Wiberg bond indices (WBI) and The topological properties calculated at bond critical points o f M − L bonds in ML (M = Co, Ni and Cu) complexes at the BP86/ def2-TZVP level of theory.
M…L Bond	M…L Bond Length	NBO			QTAIM
M…L Bond	M…L Bond Length	qM	qL	WBI	\(\rho \left({r}_{c}\right)\)	\({\nabla }^{2}\rho \left({r}_{c}\right)\)	-Gc/Vc
Co…O1	2.00825	0.87993	-0.61831	0.3509	0.0769	0.3702	-0.8759
Co…N1	2.11821		-0.44971	0.2775	0.0679	0.2716	-0.8558
Co…N2	2.35270		-0.39086	0.1427	0.0436	0.1468	-0.8744
Ni…O1	1.87510	0.59715	-0.53645	0.5119	0.1023	0.5205	-0.8639
Ni…N1	1.91793		-0.39071	0.4710	0.1040	0.4284	-0.8224
Ni…N2	2.98149		-0.39311	0.0192	0.0152	0.0380	-0.9250
Cu…O1	1.96688	0.89204	-0.60467	0.3310	0.0834	0.3834	-0.8498
Cu…N1	2.01885		-0.45849	0.2942	0.0836	0.3089	-0.8151
Cu…N2	2.89248		-0.39400	0.0283	0.0165	0.0419	-0.9490

3. 3. Cytotoxicity In Vitro

MTT assay was used to analyze the cytotoxicity of the synthesized compounds against lung (A549) and gastric (AGS) cell lines. The reduction of the yellow MTT by the mitochondrial dehydrogenase’s enzymatic activity was followed by analyzing the cell viability. Figure 4 illustrates the percentage of the cell viability of the comparative cytotoxicity patterns The IC₅₀ values of the complexes, the amount of material that kills 50% of the cells, was measured from the dose-survival curves for the growth inhibition of the two cell lines (Table 5). We found out high efficiency in the Cu-L stopping the cell lines’ growth. In addition, comparing IC50 values helps us to find out that AGS cells (average IC50 value; 6.48 µM) are more chemosensitive than A549 cell (average IC50 value; 25.46 µM). Finally, extreme cytotoxicity in the Cu-L (average IC50 values < 10 µM) –was greater than other complexes. Accordingly, our sole concern in this study was to analyze the compounds’cytotoxic potency on the breast (AGS) cell line. IC50 values of 5.01 (for Cu-L) illustrated its greater toxicity properties than other complexes. As the first cause of death all through the world, lung cancer causes 1.6 million deaths and ~ 1.8 million new cases each year. Having in mind the above considerations, Cu-L (IC50 value; 13.86 µM) illustrated more toxicity for (A549) cell line [ 50–51].

Table.5. Comparison of cytotoxicity (IC₅₀ values based on µM)

Structure	AGS	A549
Ni-L	7.29 ± 0.24	29.32 ± 0.6
Cu-L	5.01 ± 0.19	13.86 ± 0.91
Co-L	7.14 ± 0.21	33.21 ± 1.02

3.4. Antioxidant property:

The antioxidant proficiency of the synthesized compounds was studied by carrying out the DPPH• (2,2-diphenyl-2-picryl-hydrazyl) free radical scavenging technique and the results were reported in terms of radical scavenging percentage and IC₅₀ values. Table 6 shows the antioxidant activity of all complexes along with standards, ascorbic acid, and quercetin. It can be seen that the compounds displayed moderate to significant antioxidant activity ranging from 36.6 to 79.7% with IC₅₀ values of 0.33 to 0.56 mg.ml^− 1 which were less than the standards ascorbic acid and quercetin. The effectiveness of the samples as DPPH radical scavengers were in the order: Cu (IC₅₀ = 0.33 mg.ml^− 1) > Ni (IC₅₀ = 0.39 mg.ml^− 1) > Co (IC₅₀ = 0.56 mg.ml^− 1) indicating that the presence of Cu in Schiff base complexes can be effective in producing antioxidant activity in lower concentrations.

Table 6

The Antioxidant activity of the synthesized compounds and two standards (ascorbic acid and quercetin)
Sample	Concentration (mg.ml^− 1)					Average	IC₅₀
Sample	0.2	0.4	0.6	0.8	1	Average	IC₅₀
Ni	39.57	51.01	56.89	69.34	78.69	59.10	0.39
Co	36.62	46.49	53.79	60.84	69.31	53.41	0.56
Cu	49.65	61.43	66.95	74.01	79.70	66.35	0.33
Ascorbic acid	74.24	75.91	79.25	81.25	84.55	79.04	0.13
Quercetin	75.26	81.36	85.75	88.44	89.29	84.02	0.13

3.5. Antibacterial activity

Antibacterial activity studies of the complexes were carried out by the in vitro disc diffusion method. All compounds were screened against two Gram-positive bacterial strains Staphylococcus aureus (S. aureus), Bacillus subtilis (B. subtilis), and two Gram-negative bacteria strain Escherichia coli (E. coli), and klebsiella oxytoca (K. oxytoca). The antibacterial activities of the synthesized compounds, presented in Table 7, are greater against the Gram-negative bacteria than towards the Gram-positive ones. This behavior can be attributed to the structures of bacteria cell wall which results in different rates of diffusion of the compounds into the cells. A large part of cell walls in the Gram-positive bacrteria consists of thick layers of peptidoglycan, which is a more rigid structure than the Gram-negative bacteria which has a much thinner layer of peptidoglycan. Based on the obtained results, the Cu-complex (with average inhibition zone: 24.8 mm) has the most effect on both Gram-negative and -positive bacteria. B. subtilis (+) showed the most resistance, and K. oxytoca (-) was the most sensitive bacteria to the synthesized compounds. Compared to the reference antibiotics the antibacterial activities of synthesized compounds show significant inhibitory potency against the tested bacteria and that the Cu complex has shown greater antibacterial activity than the other complexes.

Table 7

Antibacterial activity of synthesized compounds and four antibiotics against selected bacterial strains.
Sample	Inhibition zone (mm)
Sample	B.subtilis	S.aureus	E.coli	k. oxytoca
Ni	17	19.3	22.5	24.3
Co	14.5	17	19	22.5
Cu	22	23.8	25.8	27.5
Penicillin	13	13	18	15
Ampicillin	14	16	12	14
Vancomycine	17	15	23	21
Tetracycline	21	22	27	23
Methanol	Na	Na	Na	Na

We have prepared a new hexadentate N₄O₂ Schiff-base ligand (L) derived from the condensation of 1,4-bis(2-Aminophenyl)piperazine with 2-hydroxy-benzaldehyde and prepared the Ni (II), Cu (II) and Co (II) complexes the direct reaction of the ligand with the metal perchlorates. The ligand and complexes were characterized by IR and Mass spectroscopy, elemental analysis and by the determination of the single crystal X-ray structure of [CoL]. In this complex, the cobalt is in a distorted trigonal prismatic coordination environment, surrounded by the six donor atoms of the deprotonated hexadentate ligand. The geometries of [ML] (M = Co, Ni and Cu) complexes were fully optimized using the B3lyp and BP86 levels of theory with def2-TZVP basis set. The results show that all three metal complexes have distorted Trigonal-Prismatic coordination geometries, similar to the crystal structure of [CoL]. The nature of the M←O and M←N bonds in the ML (M = Co, Ni and Cu) complexes were determined by QTAIM and NBO analyses. According to the NBO analysis, the maximum values of the Wiberg bond indices and charge transfer are attributed to the M←O and M←N1 bonds in NiL complex. The topological properties that were calculated confirm the covalent character of the M←O and M←N bonds in the [ML] complexes. (CuL) illustrated its greater toxicity properties than other complexes. The antibacterial studies have shown that the complex [CuL] has the greatest activity, on both on Gram-negative and Gram-positive bacteria, with E. coli (-) being the most sensitive bacteria to [CuL] while S. aureus (+) was the most resistant bacteria. The antioxidant activity of the synthesized compounds showed that, at high concentrations, the complex [CuL], had a similar scavenging activity to quercetin. Also [CuL] illustrated its greater toxicity properties than other complexes.

Data availability

CCDC 2109477 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/structures.

Acknowledgements

We are grateful to the Faculty of Chemistry of Bu Ali Sina University, for financial support. This research was undertaken, in part, using the MX2 beamline at the Australian Synchrotron, part of ANSTO, Victoria, Australia, and made use of the Australian Cancer Research Foundation (ACRF) detector.

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Synthesis of three new macroacyclic Schiff base complexes and the investigation of their anticancer, antioxidant and antibacterial properties: theoretical and solid state structures

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Abstract

Figures

1. Introduction

2. Experimental

2.1. Materials

2.2. Physical measurements

2.3. X-ray crystal structure determination

2.4. Computational methods

2.5. Cytotoxicity in vitro

2.6. Antioxidant property

2.7. Antibacterial activity

2.8. Synthesis

3. Result And Discussion

3.1. X-ray structure

3.2. Theoretical studies

3. 3. Cytotoxicity In Vitro

3.4. Antioxidant property:

3.5. Antibacterial activity

4. Conclusions

Declarations

References

Additional Declarations

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