SYNTHESIS, BIOLOGICAL STUDY, DNA INTERACTION OF MANNICH BASE METAL COMPLEXES DERIVED FROM BENZAMIDE

Objective: Coordination compounds occur widely in nature and they comprise a large share of current inorganic research. The Mannich reaction is a classic method for the preparation of Mannich bases, namely, β amino compounds, which are heterocyclic. 
Methods: A novel Mannich base of N-((3,4-dimethoxyphenyl)(2,5-dioxopyrrolidin-1-yl) methyl)benzamide and its coordination complexes with transition metals Mn and Co have been synthesized and characterized by elemental analysis, molar conductance, magnetic susceptibility measurements, UV-Visible, IR, NMR, and Mass spectral studies. 
Results: Based on the magnetic moment and UV-Visible spectral data, octahedral geometries were assigned for the metal complexes. The metal complexes were screened for antifungal activity. 
Conclusion: the metal complexes have shown good activity than the ligand. The binding of selected metal complexes with calf thymus DNA was investigated. It is found that the cobalt (II) metal complex of the ligand showed efficient DNA binding ability.


INTRODUCTION
Transition metals have an important role in medicinal biochemistry. Research has shown significant progress in the utilization of transition metal complexes as drugs to treat several diseases like carcinomas, lymphomas, infection control, anti-inflammatory, diabetes, and neurological disorders [1][2][3][4]. Manganese is an essential element involved in many chemical processes in the body, including the processing of cholesterol, carbohydrates, and protein. Iron compounds include hemoglobin which keeps our blood red. Iron atoms also help to join organic molecules, forming bactericides or bacteriostatic agents [5]. Cobalt contained in Vitamin B12 is important in protein formation and DNA regulation. Cobalt-60, a radioactive isotope, is used as a commercial source of high-energy radiation in medicine to destroy cancerous tissue. Cobalt-containing drugs are used as cyanide antidotes. Mannich bases of benzamide and succinimide are known to play a prominent role in medicinal chemistry. Despite the assets of available paper, it is found that they possess various pharmacological properties as hypnotic and tranquilizer activity, CNS depressant and cardiac stimulant, tumorinhibiting properties, and antibacterial activity [6][7][8]. Moreover, the presence of Mannich side chain increases the solubility and bioavailability of the drug molecule [9][10][11]. This work focuses on the synthesis of Mn(II) and Co(II) complexes with a new series of Mannich bases derived from succinimide, benzaldehyde, and benzamide.

MATERIALS AND METHODS
All the reagents and solvents used for the synthesis of ligand and the metal complexes were AR grade quality and have been utilized with no purification. Elemental analysis was performed using Carbo Erba 1108 analyzer and Coleman N analyzer and was found within±0.5%. The molar conductivities of metal complexes were measured in approximately 10-3 M ethanol solution using a systronics direct reading digital conductivity meter-304 with dip-type conductivity cell. The IR spectra were recorded as KBr pellets on Perkin-Elmer 1000 unit instrument. Absorbance in the UV-Visible region was recorded in DMF solution using a UV-Visible spectrometer. The 1H and 13CNMR of the ligand were recorded in the Bruker instrument employing TMS as internal standard and DMSO-DMF as internal solvent. Magnetic susceptibility measurements at room temperature were made by using a Guoy magnetic balance. Anti-microbial screening of the test compounds was carried out using the Agar-well diffusion method.

Preparation of N-((3,4-dimethoxyphenyl)(2,5-dioxopyrrolidin-1-yl) methyl)benzamide (NDB)
The new Mannich base N-((3,4-dimethoxyphenyl)(2,5dioxopyrrolidin-1-yl) methyl)benzamide (NDB)has been synthesized by the condensation of equimolar quantities of succinimide, dimethoxy benzaldehyde, and benzamide by the following method. 1g (0.01 mol) of succinimide and 0.48g (0.01 mol) of benzamide was dissolved in a small amount of distilled water and taken in a 100 ml beaker. 1.67g (0.01 mol) of dimethoxy benzaldehyde dissolved in acetone was added dropwise to the beaker solution and stirred with a magnetic stirrer for one day. Then it is kept aside with occasional stirring for few days. After a week a solid product formed was filtered, washed with distilled water and dried in an air oven at 60 ⁰C, and recrystallized using ethanol and CHCl3 in 1:1 ratio ( fig. 1).

Synthesis of manganese sulphate complex of NDB
To a salt solution of Manganese sulphate (0.05g), ethanolic solution of the ligand NDB (0.1g) was added slowly with stirring. The solution is stirred well in a hot late with a magnetic stirrer to get a clear solution. It is covered with aluminum foil and kept aside for 2 w. After slow evaporation of the solvent at 28 ⁰C, a dull white colored compound was obtained. It was filtered and dried in a vacuum ( fig. 2).

Synthesis of manganese chloride complex of NDB
To a solution of Manganese chloride (0.061g), ethanolic solution of the ligand NDB (0.1g) was added slowly with stirring. After stirring for 4 h, a clear solution was obtained. It is covered with aluminum foil and kept for a few days. A resulting dull-white solid was separated on evaporating the solution at room temperature. It is filtered and dried in a vacuum ( fig. 2).

Synthesis of cobalt chloride complex of NDB
The cobalt chloride complex of NDB was prepared by refluxing a hot suspension of cobalt chloride (0.735g) with an ethanolic solution of the ligand (0.1g) for an hour to obtain a clear solution. Light blue crystals of the metal complex were formed by the slow evaporation of solvent after 2 w. The compound was filtered and dried in a vacuum ( fig. 2).

Physical measurements
The physical properties and elemental analysis of the prepared ligand and its metal complexes are described in table 1 and 2. The structures of the metal complexes were further confirmed by conductivity measurements and magnetic moment determinations. The molar conductance studies reveal that all the complexes are non-electrolytes. The CHNO values are also in agreement with the calculated values.

UV-visible spectroscopic measurements
Absorbance in the UV-Visible region was recorded in DMF solution using the UV-Visible spectrometer (table 3).

FT-IR spectral analysis of the ligand NDB and its metal complexes
For finding out the possible coordination sites, the IR spectra of the ligand and metal complexes were recorded in FT-IR spectrometer in the KBr phase. Characteristic vibrational bands of the ligand and its metal complexes and their assignments are given in table 4. The IR spectrum of the free ligand exhibited a strong band at 1680 cm-1 for ν(C=O) stretching of the succinimide ring [16]. A band at 3302 cm-1 corresponds to the stretching vibration of the (N-H) bond. In metal complexes, there is a shift of ν(C=O) vibration to lower frequency range suggesting the coordination of carbonyl group of succinimide ring with the metal atoms in the complexes. The N-C-N stretching frequency of the ligand at 1400 cm-1 was lowered in the metal complexes confirming the involvement of the nitrogen atom of benzamide with the metal atoms. This is further supported by the appearance of new bands around 400-420 cm -1 in the far IR region for the coordination of metal-nitrogen and metal-oxygen [17].

H NMR spectral data of NDB
The 1 H NMR spectra of NDB shows a singlet at δ 2.57 due to methylene proton. The doublet at δ 3.35 δ is assigned to methoxy proton. A multiplet between 6.26-6.88 δ is assigned for aromatic protons. The singlet for one proton at δ 8.48 is assigned to-NH proton.

C NMR spectral data of NDB
The number of signals of sharp peaks represents the number of carbons of the ligand, which are not chemically equivalent. 13

Mass spectral data of NDB
The molecular formula of NDB based on elemental analysis is C20H20N2O5. Observed molecular mass by LC-MS spectrometer is 367.25, which is in closer value with the calculated value 368.14.

Anti-fungal activity
Mannich bases and their metal complexes have potent antimicrobial activities [19]. The anti-fungal property of the synthesized Mannich base and its metal complexes were examined against A. niger and C. albicans. The anti-fungal activity of each compound was compared with the standard drug Fluconazole. The inhibition zones were measured at the concentration of 100µg/ml and 400µg/ml. at the concentration of 400µg/ml, all the metal complexes have shown better anti-fungal activity against the selected microorganisms. The percentage inhibition for fungi is calculated after five days using the formula.
Percentage of inhibition = 100(X-Y)/X (X-Area of the colony in control plate; Y= Area of the colony in test plate).

DNA binding and cleavage activities
The interaction of the synthesized Co (II)sulphate complex with DNA is monitored with the help of absorption titrations. At fixed concentrations of the complex, the interaction with DNA (pBR322 and calf thymus) was investigated using UV absorption spectra. There is a redshift (hypochromic effect) in the absorption spectra for the metal complex, which is usually characteristic for non-covalent intercalative binding of the compound to DNA helix, which is because of effective stacking interaction of aromatic chromophore of the compound with the base pairs of DNA [20]. Fig. 3 represents the absorption spectra of the complex in the absence and presence of increasing amounts of DNA. In the UV region, a strong absorption peak was observed due to the metal complex. The gradual addition of DNA resulted in hypochromism and bathochromic shift in UV spectra [21]. The bathochromism result is due to the decrease in the energy of π-π* transition. Here, the π orbital of the DNA base pair might have been coupled with π-π* orbital of the intercalated ligand.  . 4). But at 50µM concentration, the complex has cleaved DNA completely [23]. 40µM H2O2 alone has not displayed any activity over DNA.

ACKNOWLEDGEMENT
The author wishes to thank Madras University for DNA binding studies, Biogenics Hubli for DNA cleavage studies and IIISM, SRMIST for spectral studies.