Introduction

Heterocyclic compounds represent a large family of organic compounds (Brimble, Gibson, & Sperry, 2008; Katritzky, Ramsden, Joule, & Zhdankin, 2010), they are known for widespread applications in various fields because of their biological importance, medicinal chemistry, and chemical biology. The 2H-chromene (2H-1-benzopyran) moiety is one of the most important heterocycles detected in many biologically active natural products and in synthetic compounds (Ellis, 2009; Kolokythas et al., 2007; Schweizer & Meeder, 1977) 2H-chromenes were reported to display significant antioxidant (Kwak et al., 2006), antiviral (Martínez-Grau & Marco, 1997), antimicrobial (Li et al., 2018) anti-inflammatory (Gebhardt et al., 2007) and antifungal (Chanu et al., 2017) properties, in addition to their interesting applications as highly effective fluorescent dyes (Moorthy, Venkatakrishnan, Sengupta, & Baidya, 2006) Furthermore, several metal complexes of chromene derivatives possess antimicrobial (Soliman, Mohamed, & Elgemeie, 2016) DNA cleavage activities (Prabhakara et al., 2015) and photoluminescence properties (Aazam, Husseiny, & Al-Amri, 2012) . Azo compounds and their complexes are highly important due to their applications as textile dyes, (Zhigang et al., 2009) indicators (Abbas et al., 2018), medicinal importance (Philip, Thomas, & Mohanan, 2019) and are also inhibition of DNA, RNA and protein creation (Park et al., 2007).

In the present study, we display the synthesis and characterization of Co(II), Cu(II), and Ni(II) complexes with new azo- linked chromene ligand (AH). The newly ligand and its complexes were examined for their antibacterial potency against some types of gram-positive and negative bacteria; DNA cleavage activity, and also fluorescence properties.

Materials and Methods

All chemicals were from BDH, Merck, and Fluka products. ¹H and ¹³C–NMR spectra were performed using Burker 500.13 MHz. Elemental analyses were carried through a Perkin CHN Elmer analyzer. The metal content was determined by varian-AA6200 flame atomic absorption spectrophotometer. Gallenkamp M.P. apparatus were used. Molar conductance of the prepared complexes with 10⁻³M in EtOH were measurement using SIEMENS conductivity meter. Magnetic susceptibility measurements were obtained using the MSB-MK-1 model. The UV-Vis spectra were obtained with SHIMADZU-1800 spectrophotometer using a quartz cell (200-1100 nm). The IR spectra were measured using SHIMADZU FTIR-8400S spectrophotometer using (KBr & CsI discs). The fluorescence measurements of the prepared compounds were recorded on a SHIMADZU Fluorescence instrument.

Synthesis of The Starting Material

Synthesis of chalcone compound (B) [ 1-(2-hydroxyphenyl)-3-(4-nitrophenyl)prop-2-en-1-one ]

A mixture of p-nitrobenzaldehyde (10 mmol), 2'-hydroxyacetophenone (10 mmol) were dissolved in (25 ml) methanol, Potassium hydroxide (30 mmol) was added with stirring at room temperature for 10 h to obtain a blood red mixture, then infused into 1N HCl (20 ml), and concentrated HCl was added until the solution was acidic. The precipitate was filtrated, washed with water and recrystallized from methanol. C₁₅H₁₁NO₄ (269 g.mol^-1), Light orange powder,yield: 73%, m.p.: 210-212 ⁰C. FTIR (KBr, cm^-1): 3548 (-OH group), 1639 (C=O), 1267 (C-O, phenolic). ¹H-NMR (500MHz, DMSO-d₆, δppm) δ 11.84 (s, 1H), 8.31-8.17 (m, 2H), 7.93-7.91 (m, 1H), 7.72 (d, J=10Hz, 2H), 7.54-7.50 (m,1H), 7.05-6.93 (m, 1H); ¹³C-NMR (125 MHz, C₆D₆, δ ppm) δ 203.6 (C=O), 160.9 (C-O), 153.5 (C-N), 146.9 (C), 141.3 (C), 131.4 (C), 127.7 (C), 127.5 (C), 123.8 (C), 123.7 (C), 121.3 (C), 119.6 (C), 118.0 (C), 99.9, 68.8 (CH=CH).

Synthesis of 2H-Chromene Azo Ligand (AH) 2-(4-nitrophenyl)-N-(4-(phenyldiazenyl) -2H-Chromen-4-Amine

A mixture of the prepared chalcone compound (1.67 mmol, 0.44 g) and p-amino azobenzene (3.34 mmol, 1.49 g) was refluxed in absolute ethanol (50 ml) with concentrated hydrochloric acid for 30 h. The precipitate was filtered and washed with the hot solvent; Figure 1. C₂₇H₂₀N₄O₃ (448 g.mol^-1), Dark purple solid yield: 85 %, m.p.: 228-230 ⁰C, FTIR (KBr, cm^-1): 3485 (N-H), 3041 (aromatic C-H), 1658 (aromatic C=C), 1452 (N=N). ¹H-NMR (500 MHz, DMSO-d₆, δ ppm) δ 9.23 (s, NH), 7.91 (d, J=5Hz, 1H), 7.85 (d, J=10Hz, 1H), 7.54 (dt, J=15, J=7.5 Hz, 1H), 7.33 (d, J=10Hz,1H), 3.44(q, J=6.6Hz, 1H), 2.07 & 1.05 (s, t, J=7.5Hz, CH, CH); ¹³C-NMR (125 MHz,C₆D₆, δ ppm) δ 159.8 (C-O), 151.9 (C-N), 148.9 (C-N), 148.2 (C-N), 131.4 (C), 130.0 (C), 129.9 (C), 129.7 (C), 125.1 (C), 122.7 (C), 122.5 (C), 122.3 (C), 96.5 (C), 76.9, (C) 58.6 (C).

Synthesis of 2H-Chromene Azo Metal Complexes

To a stirred solution of 2-(4-nitrophenyl)-N-(4-(phenyldiazenyl)-2H-chromen-4-amine (AH) (0.3 mmol, 0.134 g) in (20 ml) ethanol, (0.6 mmol) of metal chloride (M= CoCl₂⋅6H₂O, CuCl₂⋅2H₂O and NiCl₂⋅6H₂O were added at room temp. The solution mixture was reflux for two hours, then allowed to cool and left overnight at room temperature. Then filtered and washed with EtOH, ethyl ether, and dried at the desiccator on CaCl₂ .

For Co(AH)(H₂O)₂Cl : pink powder; yield: 69 %, FTIR (CsI, cm^-1): 3419, 771 (H₂O coor.), 3047 (aromatic C-H), 545 (Co-O), 360 (Co-N), 316 (Co-Cl). For Cu(AH)(H₂O)₂Cl : olive green powder; yield: 74 %, FTIR (CsI, cm^-1): 3438, 840 (H₂O coor.), 3058 (aromatic C-H), 553 (Cu-O), 345 (Cu-N), 283 (Cu-Cl). For Ni(AH)(H₂O)₂Cl : Reddish pink powder; yield: 71 %, FTIR (CsI, cm^-1): 3315, 845 (H₂O coor.), 3047 (aromatic C-H), 441 (Ni-O), 356 (Ni-N), 310 (Ni-Cl).

Biological Studies

Antibacterial Studies

The biological activities (in vitro) of the prepared ligand (AH) and its chelates were performed by disc diffusion method (Bauer, Kirby, Sherris, & Turck, 1966) against two gram-positive (Bacillus subtilis, Staphylococcus aureus) and two gram-negative (Escherichia coli, Klebsiella pneumonia) using Muller Hinton agar for growing cell and the activity was measured in terms of zone of inhibition. DMSO has been applied as a negative control with ciprofloxacin (1 mg.ml^-1) as the standard drug.

DNA Cleavage Studies

The newly prepared ligand (AH) and its metal (II) complexes were analyzed as DNA cleaving agents using the bacterial previous studied DNA as targets by the agarose gel electrophoresis method (Sambrook, Fritsch, & Maniatis, 1989). Test samples (1 mg.ml^-1) were dissolved in DMSO.

Results and Discussion

The analytical results of all compounds are presented in Table 1. The elemental analysis, metal determination, and the chloride content (by Mohr's method) of the prepared complexes revealed that all complexes with 1:1 stoichiometry and the calculated values are in a good acceptance with the found values.

Biological Studies

Antibacterial Studies

The antimicrobial activity of the (AH) ligand and its metal (II) complexes were investigated against two Gram-positive (Bacillus subtilis, Staphylococcus aureus) and two Gram-negative (Escherichia coli, Klebsiella pneumoniae). The inhibition zones were determined and compared with ciprofloxacin (standard drug), as shown in Figure 2. The results indicate that the ligand (AH) and Co(II) complex has no effectiveness versus Staphylococcus aureus, but, the Cu(II) complex has more inhibition than the Ni(II) complex against this bacteria. The promoter activity of these metal complexes over the ligand can be elucidated on the principle of chelation theory (Chohan, Arif, Akhtar, & Supuran, 2006; Sharma & Chandra, 2011). In chelating, the positive charge of the metal ion is partial participate with the donator group present in the ligand and probable π-electron delocalization upon chelated mode (El-Wahab, Mashaly, Salman, El-Shetary, & Faheim, 2004).

Ni(II) complex has no activity against Escherichia coli, Klebsiella pneumoniae and Bacillus subtilis bacteria, whereas Co(II) complex has just action against Bacillus subtilis, the prepared compounds showed variable effectiveness against different organisms which refers either on the impermeability into microbes cells or on variance in ribosome of microbial cells (İspir, 2009).

DNA cleavage studies

The DNA cleavage activity of the new ligand AH and its complexes have been studied against DNA of Staphylococcus aureus, from the results of DNA cleavage experiment as shown in Figure 3, it was noticed that the DNA control (untreated DNA) does not exhibit any cleaving and there was a difference in the migration of ligand alone and its chelates. This damage to DNA in AH ligand, Co(II), Cu(II) and Ni(II) complexes could be attributed to the cleavage of DNA. It was observed that the lane Cu(II) complex showed complete DNA cleavage of Staphylococcus aureus, but the free ligand, Co(II), and Ni(II) complexes showed partial cleavage of DNA. These data refer to the significant property of coordination to metal ion (Karekal, Biradar, & Mathada, 2013; Mahalakshmi & Rajavel, 2014).

Many previous studies infer that the compound was to split the DNA; it can prohibit the evolution of the pathogenic organism via the genome cleaving (Yousef, El-Reash, El-Gammal, & Bedier, 2013).

Fluorescence studies

The fluorescence properties of newly synthesized 2H-chromene azo ligand and its complexes were measured in (10^-5 M) Ethanol and DMSO solutions. The fluorescent spectra of the free ligand and its complexes when excited at 387 nm in Ethanol, showed a maxima band at 425 nm, as in Figure 4. The low intensity of the band corresponding to complexes is due to a decrease in electron density on the ligand. The low band intensity that belongs to complexes caused the decreasing electron density of the ligand (Yamgar, Sawant, Sawant, & Chavan, 2009).

The free ligand exhibits an emission maximum at 569 nm with excited at 530 nm in DMSO, and the Cu(II) complex noted an emission band at 565 nm as in Figure 5: but, at the same excited band (530 nm), the Co(II) and Ni(II) complexes possess no fluorescence. The chelation of the metal with ligand performing the posing of the nonfluorescent complex; consequently, the fluorescent intensity is decreased and promotes quenching (Philip et al., 2019)

NMR Studies

The ¹³C-NMR spectrum of the 2'-hydroxy chalcone (B) shows a significant signal at 203.6 ppm corresponding to carbonyl group that was absent in the spectrum of the 2H-chromene azo ligand.

The ¹H-NMR spectrum of the 2'-hydroxy chalcone (B), signal at 11.84 ppm was referred to the phenolic (–OH) proton, while in the spectrum of 2H-chromene azo ligand (AH), the signal at 9.23 ppm (s, 1H, NH) is due to NH proton (El-Deeb, Ryu, & Lee, 2008), the disappearance of this signal in all spectra of metal complexes indicate its involvement in complexation with metal ion through nitrogen atom via deprotonation.

Infrared spectral studies

The IR spectrum of the prepared starting material 2'-hydroxy chalcone shows the characteristic band at 3548 cm⁻¹ indicated that the presence of hydroxyl group and 1639 cm^–1 remarked the α, β unsaturated carbonyl (>C=O) (Sumathi, Tharmaraj, Sheela, & Ebenezer, 2011).

In IR spectrum of 2H-chromene azo ligand (AH) exhibited significant features, the peak at 3485 cm^-1 was assigned to the secondary (NH) vibration of the ligand which was absent in all-metal complexes, due to deprotonation of (NH) group on binding with metal ions, while the band at 3045 cm^-1 was characteristic for the existing of aromatic (-C-H) stretching. No shift group was observed in the stretching vibration of the azo group (N=N) of the ligand. This supports the non-involved of an azo group in the metal coordination (Tunçel & Serin, 2006).

The appearance of lower frequencies due to bands of metal bonds with nitrogen, oxygen, and chloride in all metal complexes and show other bands about the region (3438-3315 cm^-1) and at the range (845-771 cm^-1) ascribed to coordinated water (Aazam et al., 2012).

Electronic Spectra, Magnetic Susceptibility , and Molar Conductivity Measurements

The electronic spectra of ligand and all prepared complexes were recorded in DMSO at r.t.; in the spectra of free ligand showed two absorption bands. The first at 255 nm attributed to π–π* transition of the benzene ring (Khanmohammadi & Darvishpour, 2009; Khedr, Gaber, Issa, & Erten, 2005). The second band noticed at 326 nm can be attributed to n–π* transition. An azo of free ligand (403 nm) did not note a significant shift in all the complexes, it was considered as evidence that no participation of the azo group in complex formation. The Co(II) complex showed two bands at 677 and 615 nm, which can be assigned to the d-d transitions [⁴A₂(F)→ ⁴T₁(F) & ⁴A₂(F)→ ⁴T₁(P) ]. These bands, with the magnetic moment of the complex (µ = 4.65 B.M.), usually correspond to a tetrahedral environment around Co(II) ion (Knittl, Abou-Hussein, & Linert, 2018; Lechat, Khan, Bouet, & Vierling, 1993). The electronic spectrum of Cu(II) complex exhibited band at 989 nm and other at 891 nm that might be assigned to ²B₂→²E and ²B₂→²B₁(²A₁) transitions, indicating the Cu(II) complex has distorted tetrahedral geometry (Lever, 1984; Pahonțu et al., 2015) with the magnetic moment (µ = 1.89 B.M.). In the Ni(II) complex, the electronic spectrum showed bands at 646 nm and 785 nm; these bands may be attributed to ³A₂(F) → ³T₂(F) and ³A₂(F) →³T₁(F) transitions, respectively. It suggests a tetrahedral geometry of the Ni(II) complex (Lever et al., 1984), and its the magnetic susceptibility equals 3.31 B.M., which sited at the typical field for a tetrahedron geometry.Figure 6 showed the proposed structure of all metal complexes. Molar conductance of the complexes was recorded in ethanol (10^-3 M). The lower value of molar conductance for all prepared complexes indicates their non-electrolytic character (Geary, 1971), as shown in Table 2.

Conclusion

In this study, the 2H-Chromene azo ligand and its chelates with Cobalt(II), Copper(II) and Nickel(II) ions have been synthesized and characterized using various analytical and spectral data. The results demonstrate that the ligand is monodentate; all metal chelates exhibited non-electrolyte and tetrahedral geometry. Antibacterial activities of the free ligand and its metal complexes were investigated against gram (+) and Gram (-) bacteria strains and revealed that Cu(II) and Ni(II) complexes were effective comparables with free ligand against S. aureus bacteria. Cu(II) complex activity upon the DNA cleavage studies showed more effective against the DNA isolated from S. aureus bacteria than the free ligand and other its metal chelates.