SYNTHESIS, CHARACTERIZATION, POM ANALYSES AND BIOLOGICAL EVALUATION OF N-[(2-METHOXY-5-NITROPHENYL)]-4-OXO-4-[OXY] BUTENAMIDE BASED ZINC(II) CARBOXYLATE COMPLEXES

The aim of medicinal chemistry is to links many scientific disciplines and allows the scientists in researching and developing new drugs with enhance and targeted properties. In this article we are exploring the preparation of four new zinc(II) carboxylate complexes based on N-[(2-methoxy-5-nitrophenyl)]-4-oxo-4[oxy]butenamide which were characterized through FT-IR and EDX studies. The DNA binding ability and binding type of complexes were assessed by spectroscopic (UV-Visible) and viscosity measurements, exhibiting an intercalative pattern of interaction. The synthesized compounds were also assessed to know theoretically about their nature by molecular docking studies resulting also in intercalation mode. Analysis of the complexes for biological applications such as anti-microbial, anti-leishmanial, cytotoxicity and DNA damage activities showed that these complexes carries good anti-microbial, anti-leishmanial activity with no toxicity to human blood thyrocytes and DNA. The bioavailability prediction and drug likeness score has also been evaluated through Insilco studies.


INTRODUCTION
Zinc has got importance because of its catalytic, enhanced biological activities of ligand along with highly inhibiting activities against fungal and bacterial growth [1][2][3]. The amide-based carboxylate ligands were designed for the current study because of the existence of peptide bonds in its structures which is highly important on the biological side because of its larger hydrophilic character as compared to the simple carboxylates [4,5]. The carboxylate complexes of zinc(II) with ligands of nitrogen or oxygen donors have been proved highly significant in biochemical aspects, i.e. biological and chemical point of view in current era [6,7].
In coordination chemistry, Zn is an attractive central metal ion and is an essential part of all enzyme classes. Zinc performs diverse physiological functions in many biological processes, for example, as a structural component, as a catalytic factor, or as a signaling mediator [8]. Curcuminate based zinc(II) complexes having an aromatic spectator ligands displayed in vitro anticancer activity against various cancer cell lines [9]. Some Zn(II) carboxylate complexes have shown excellent anti-diabetic activity as Zn plays a vital role in insulin storage and secretion, as well as its reported insulin mimetic properties [10]. Zn(II) complexes have also displayed antitumor activity against human cancer cell lines because of the cytoprotective nature of Zn and suppresses apoptotic pathways [11]. Amide based Zn(II) carboxylate complexes have also been used for Alzheimer's disease treatment against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) [12].
New drugs with better cure rates and minimum side effects are required for the cancer treatment. Coordination chemistry presents lot of compounds having various geometries, different redox reactivity and different modes of attachment to DNA, e.g. cis-platin and other platinum coordination compounds are among the most widely used drugs for cancer treatment. Due to some sever side effect of Pt based drugs, coordination compounds of other than Pt metals with good antitumor activity got the attention of the researcher for the development of new anticancer agents [13]. Zinc(II) carboxylates possess various mode of interaction with DNA like intercalation, grove binding and electrostatic interaction [14][15][16].
The aim of this research work is to prepare N-[ (2-methoxy-5-nitrophenyl)]-4-oxo-4-[oxy]butenamide based complexes of Zn with oxygen/nitrogen donor ligands capable of exploiting the antimicrobial, anti-leishmanial activities and carries lesser toxicity to human blood erythrocytes and DNA.

Materials and methods
Solvents and other required chemicals of analytical grade purity were obtained from Sigma and Alfa Aesar and used as such as received. A digital Electro-thermal melting point instrument was used to determine the melting points of the prepared samples. FTIR spectra in the range of 4000-400 cm -1 of prepared compounds were obtained using Thermo fisher FTIR spectrophotometer. The DNA binding interaction was validated through UV-Visible spectrophotometer. EDX instrument was employed to determine the elemental compositions of the prepared compounds.

Synthesis of N-[(2-methoxy-5-nitrophenyl)]-4-oxo-4-[oxy]butenamide (HL) and NaL
The ligand HL was re-synthesized by dissolving in glacial acetic acid separately maleic anhydride and 2-methoxy-5-nitroaniline and then mixed them slowly. The mixture was stirred at 25 ºC and precipitate formed after few min. The resulting precipitate was filtered and washed thoroughly using H 2 O to get the desired product after air dry [17].
Sodium salt (NaL) was obtained by following the procedure described by Sirajuddin and coworker [17] by the slow addition of NaHCO 3(aq) to a flask containing suspended solution of ligand, HL in distilled H 2 O. After few min stirring at 25 ºC, a clear solution was achieved which gives the required NaL product after rotary evaporation of H 2 O (Scheme 1).

Synthesis of bis(N-(2-methoxy-5-nitrophenylamino)-4-oxo-4-[oxy]butenamide)zinc(II) (1)
Complex 1 was obtained by reacting an aqueous solutions of NaL (4 mmol, 1.16 g) and zinc sulfate (2 mmol, 0.574 g) in 2:1 stoichiometric ratio at room temperature [18] as presented in Scheme 1. Precipitate of the required complex 1 was appeared after stirring the resulting mixture at 25 ºC. Precipitates of the required complex 1 was filtered, washed with distilled H 2 O and dried. The desired complex was tried for crystallization in different solvents like methanol and DMSO but failed to get good quality crystals.

Complex-DNA binding study via UV-Visible spectroscopy
The deoxyribonucleic acid solution was obtained by the dissolution of small amount of sodium salt of SS-DNA in distilled water that was kept on stirring at room temperature for overnight to get the clear homogeneous solution. The ratio of n/p (nucleotide to protein) ratio for the prepared solution was obtained from the absorbance ratio of A 260 /A 280 and was about of 1.9 which indicates that DNA is free from protein. The concentration of DNA was calculated to be 1.97 x 10 -4 M via using the molar absorptivity value 6600 M -1 cm -1 at 260 nm [19][20][21]. The DNA solution was stored at 4 ºC. Complex solutions (20 µM) were prepared and screened for DNAbinding studies. The concentrations of complexes were kept constant while that of the SS-DNA was varied during the experiment. To both the complex and reference cell, an same quantities of DNA concentration were added to get variable absorbance [22,23].

Complex-DNA binding study via viscometry
The viscosity of complex-DNA adduct was measured using the Ubbelohde Viscometer at room temperature to know the binding behavior of the complex-DNA interaction. In order to record mean flow time the process was done in triplicate. The data were plotted between the relative viscosity and the ratio of the complex-DNA concentration, i.e. (η/η o ) 1/3 vs.
Here η is the viscosity in the presence of the complex and η o signifies the viscosity of the DNA in the absence of the complex [24].

Anti-bacterial activity assay
The anti-bacterial activity of the screened compounds against five bacterial strains (Klebsila pneumonia, Streptococcus auras and Escherichia coli were determined using well diffusion assay. All the experimental materials and nutrient agar media were sterilized while autoclaved for twenty min at 121 ºC. Zinc(II) carboxylates complexes of 1 mg/mL to 6 mg/mL were prepared in DMSO. Azithromycin and DMSO were used +ve and -ve controls, respectively. A saline solution of 0.49 g NaCl in 50 mL distilled water was also prepared. A homogeneous solution of Stock nutrient Agar was obtained by dissolving 5.5 g of it taken using digital balance and was solubilized in 200 mL distilled H 2 O [25][26][27][28][29][30].

Anti-fungal activity assay
Different dilutions of the screened samples were prepared having concentrations 1-6 mg/mL in DMSO. Respective solution of Turbinofine was also prepared in DMSO which was used as +ve control. Sabouraud dextrose agar media was prepared by dissolution of 4.5 g of SDA in 80 mL distilled water. After that all experimental materials and media (SDA) were sterilized while autoclaved for twenty min at 121 ºC. The experimental activities were carried out in laminar flow which provide microbes free environment. The SDA was poured in test tubes and kept in slant position. After SDA solidification 60 µL concentration from stock solutions of each complex were added in each corresponding labeled test tubes. The media along with samples were kept for some time to get solidified. After solidification fungal strains Aspergillus niger and Aspergillus flavus were applied on each test tube with the help of wire loop. All the test tubes were closed through cotton swabs and kept in incubator at 37 ºC. The inhibition was measured once after 24 h and again after 72 h [31][32][33].

Anti-promastigote assay
The anti-promastigoate activity of the prepared compounds was performed by following the procedure described by Mehwish and co-worker [34] using MTT assay [35]. The activity of the compounds at four different concentrations (prepared in serial dilution in the range of 500-62.5 µg/mL) were accessed.

Cytotoxicity assay
The prepared compounds were tested to explore their cytotoxicity activity performed by following the procedure described earlier [34,36]. Fresh human blood was collected from volunteers and RBC were obtained by centrifugation after washing three times with PBS. A 990 µL of the remaining RBC (red blood cells) were treated with each sample (complexes 1-4) such that the final tested concentration reaches at 500, 250, 125 µg/mL followed by incubation at 37 ºC for 3 h and then the cells were centrifuged for five min at with 1000 rpm. The supernatants were collected and the released hemoglobin was confirmed at 576 nm using spectrophotometer.
Dimethyl sulfoxide and Triton X-100 (0.5%) were employed as -ve and +ve controls, respectively. With the help of Elisa plate reader, the OD was obtained at 576 nm and % hemolysis was determined by the formulashown in equation 1 [37].

DNA damage analysis (DNA laddering assay)
The DNA damage was studied by DNA laddering assay as described earlier [38]. The DNA material was extracted from fresh human blood (obtained earlier for cytotoxicity assay) using phenol/chloroform (1:1) method. A 0.5 mg/mL proteinase K was added to harvest and resuspend the cells in digestion buffer. In the presence of RNase A, the mixture was incubated at 37 ºC for 3 h and precipitated by adding sodium acetate and ice-cold ethanol to the aqueous phase followed by overnight incubation at −20 ºC and was treated with different concentrations (500 µg/mL) of selected compounds (complexes) for 3 h. The mixture was centrifuged, and pellets were collected, allowed to dry and re-suspended in tris-EDTA buffer. Using a trisacetate-EDTA running buffer on 2% agarose gel, 20 μg DNA aliquots were then electrophoresed and photographed under ultraviolet light. [39][40][41].

Theoretical study by molecular docking
Molecular docking study was performed to get the information about the interaction between the drug target and compound [42]. For this MOE-Dock program (www.chemcomp.com) was used. MOE (molecular operating environment) software was employed to draw the 3D structure of the metal-complex analogs [43]. A 3D protonated structure of all compounds was drawn which were then minimized by using the default parameters of the MOE. From the protein databank (PDB), the 3D crystal structure of the DNA (1 bna) was obtained and the tested compounds were docked into the active site of the target receptor in MOE by the default parameters as described in earlier [4] and by using MOE software the best poses were obtained after docking.

RESULTS AND DISCUSSION
The physical data including the molecular formula, mass, physical state, solubility, color and melting points of the prepared Zn(II) carboxylate complexes is given in Table 1. The complexes were freely soluble in DMSO and some also in methanol. The sharp melting points of the synthesized compounds show their purity. Table 1 presents the selected FTIR peaks observed in the synthesized compounds spectra. The deprotanation of the ligand HL was confirmed by the absence of OH peak at 3250 cm -1 . The symmetric vibration peaks in complexes are present in the range of 1337-1344 cm -1 while the asymmetric vibration peaks appears in the range of 1522-1530 cm -1 . A decrease in the value of symmetric vibration and increase in the value of asymmetric vibration was observed after complexation compared to that of the free ligand because of the attachment of oxygen of the carboxylate moiety with Zn atom [44]. The difference between asymmetric and symmetric vibrations (Δν) for complexes 1-4 are 183, 192, 188 and 182 cm -1 , receptively, indicating bidentate mode of coordination of carboxylate moiety to Zn atom as Δν for complexes <<Δν for NaL [5]. The complex formation was also confirmed by the appearance of Zn-O absorption bands at 428, 430, 424 and 420 cm −1 , respectively in complexes 1-4 [5,45]. The attachment of 2,2'-bipyridine, pyridine and 1,10-phenanthroline group in complexes was observed at 516, 470 and 479 cm −1 because of Zn-N peak [5].

EDX analysis
The prepared complexes were also characterized by EDX to study the presence of Zn. Since EDX is used mainly for the detection of heavy metals and is not suitable for the lighter elements like carbon or oxygen atoms. The presence of Zn metal in the composition confirms the complex formation as shown in Table 2. The variation is the percentage composition is due to the fact that EDX is only good for heavy metal detection.

DNA binding studies by UV-Visible spectroscopy
While studying absorption spectra for complexes one prominent peak was observed having variable wavelength and absorbance in the absence of DNA. The resultant peak appears as a result of π-π* transitions along with aromatic chromosphere in complex. After the addition of DNA solution into the complex solution, changes takes place in absorbance and wavelength, which shows the interaction between complex and DNA [46]. A hypochromic with slight red shift recorded in the spectra of the screened complexes upon the addition of DNA solution which is an indication of the intercalation type of interaction between the DNA and the compound. The π* orbital of intercalated complex molecule interact with the π orbital of the DNA bases resulting in the reduction of π-π* transition probability giving the bathochromic effect and supports the intercalation binding mode. The DNA binding constant K b (M -1 ) was obtained from the intercept to slope ratio of a plot of A o /(A-A o ) vs. 1/[DNA] [47]. The DNAcomplex interaction was stable and the binding was spontaneous which was confirmed from the -ve ∆G value. The effects for complexes 1, 2, 3 and 4 are shown in Figure 1.

DNA binding studies by viscometry
The complex binding mode to deoxy ribonucleic acid (DNA) was also confirmed using viscometer. The time flow for various concentrations was recorded. The graph for viscosity was plotted as (η/η o ) 1/3 vs. r = [Compound]/[DNA]. The intercalated binding mode of complex to DNA was confirmed from the increase in viscosity. The relative viscosity of the free DNA and in the presence of various concentration of the complexes were determined from time flow using equations 2 and 3 [48]: where t o and t are the time flow in the absence and presence of DNA, respectively. The representative plot of complex 2 with DNA shows the intercalative mode of interaction in Figure 2A.

Analysis of anti-microbial activity
The synthesized complexes were screened for their anti-bacterial potential and their activity was compared with standard antibiotic, Azithromycin. The maximum activity of 23, 22 and 24 mm were shown by Azithromycin against Klebsila pneumonia, Streptococcus auras and Escherichia coli, respectively. It was observed that the antibacterial activities were increased with increasing complex concentration ( Table 3). The minimum activity was shown by 1 mg/mL while maximum activity was shown by complex having concentration of 6 mg/mL. The complexes were evaluated after incubation period for their anti-fungal activity using Terbinafine (shows maximum inhibition) and DMSO (shows zero inhibition) as +ve and -ve controls, respectively (Table 3). It was summarized that the minimum anti-fungal activities were shown at concentration of 1 mg/mL while maximum activity shown at 6 mg/mL. The recorded data was compared with standard anti-fungal drug and negative control. It was summarized that with the increase in complex concentration increase the anti-bacterial activity as shown by various concentration results.

Anti-leishmanial activity of zinc(II) carboxylate complexes
Anti-promastigote assay of the tested compounds was performed by MTT assay using Amphotericin B as a standard and their data displayed that against L. tropica promastigote these compounds are highly active at all concentrations. Percentage inhibition of promastigote L. tropica treated with various concentrations of complex after 72 h of incubationis given in Table  4. The highest activity was shown by complexes 4 > 3 > 1 > 2. All the complexes showed over 90% inhibition at 500 μg/mL tested against promastigote. The highest activity of complex 4 may due be greater lipophilic character due to the presence of 1,10-phenthroline moiety. The data clearly indicate that dose and time dependent activity was observed for the screened compounds.

Cytotoxicity analysis
The quality of chemical and other substances being lethal to cells is called cytotoxicity for example certain immune cells and venoms [49]. The interaction of molecules with the cells (RBC's) results in the formation of membrane pores which ultimately results in the hemolysis of cell membrane by colloid osmotic mechanism to release hemoglobin. The hemolytic activity of the screened compounds was then determined spectrophotometrically by measuring the released hemoglobin. Previously this assay has been used as a reliable model for screening pharmaceutical agents designed for intracellular delivery of biologic drugs. Keeping in view the importance of the test compounds it is worth testing the possible cytotoxic effect toward human red blood cells. The percent hemolytic activity of the screened compounds explored that the screened compounds were non hemolytic to human blood erythrocytes at all tested concentrations (Table 4).

DNA damage analysis (DNA laddering assay)
DNA damage may be referred to as the ability of chemical agent that damages the genetic material inside the cell causing unexpected changes such as mutations, inappropriate event activation and other structural changes which may cause serious illness such as cancer [50]. This assay has the importance that it not only confirms degradation or damage occurring to DNA but it also gives an idea about apoptosis. Our findings suggested that these complexes have no obvious DNA laddering/smears/tailing profile when compared with the ladder and positive control H 2 O 2 ( Figure 3). Lower DNA damage activity and nontoxic naturefurther increments the significance of these complexes in the field of drug development.

POM analysis
POM physico-chemical analysis or ADME/T is important to qualify drugs and their efficacy as leading candidates against various diseases. The POM physicochemical calculations included a partition coefficient (cLogP), aqueous solubility, donor hydrogen bond and drug likeness, which are evaluated in terms of Lipinski's rule-of-five. To qualify oral bioavailability, the topological polar surface (TPSA) should be < 140 Å 2 [33]. In our compounds the value of TPSA is < 140 except only for complex 1. The presence of two different pharmacophore sites: NH−C−C−N=O (potential antibacterial pharmacophore site) and O=C−C−C−C=O (potential antifungal/antiviral pharmacophore) make the synthesized complexes quit interesting from biological point of view [47]. From molinspiration data (Table 5) it was concluded that the synthesized complexes obey the rule of Lipinski behave as a drug and to have kinase and enzyme inhibition properties. As our synthesized complex has Molecular weight less than 450, so it may be highly absorbed because most of the traded drugs, i.e. approximately 80% has Molecular weights in this range. The complex with molecular weight 595.79 also shows drug likeness as 20% traded drug has molecular weight more than 450. The logP value of the synthesized complexes fall in the standard range, i.e., less than 5 so these compounds may be highly hydrophilic and thus meet the criteria of market drugs.

Osirisis calculations
The Osiris calculations are given in Table 6. Toxicity risks and physic-chemical properties of complexes were evaluated and analyzed by the methodology developed by Osiris. It is necessary for a good drug to have high hydrophilicity and low cLogP value (cLogP value must not be greater than 5.0). It may lead to poor absorption and permeation if a drug lacks such contents. Compounds having cLogP> 5 have a good absorption ability. Upon such recommendations all the complexes exhibit cLogP values in the satisfactory range. Here 'S' shows the aqueous solubility capability of a compound. The screened compounds may be recommended to be introduce as an innovative drug because they have the 'S' value range (Table 7) which meets about 80 % of Drug market criterion (S value greater than -4). The toxicity effects of the synthesized complex were also determined through Osiris analysis which helps in determining risky fragment. All the synthesized complexes have less toxicity risk as a whole and fulfill the requirements of the drug market up to some extent.

Molecular docking studies
Molecular docking studies were carried out in order to find out the binding mode of the metal complexes and to information about the proper orientation of DNA bounded complex yielding a stable adduct. It was also confirmed by Molecular docking studies that the binding mode of complex to DNA is intercalative. In intercalation due to the introduction of an intercalator into DNA base pairs the DNA double helix arrangements may be stabilize, stiffen, lengthen and unwind. The Zn carboxylate complex analogs were docked with DNA and the results showed that the most potent inhibitor was complex 4. It was observed that this complex made one Hacceptor and two pi-H interactions with DC3 and DG4 residues of DNA respectively as shown in Figure 2B.

CONCLUSION
All the complexes were synthesized in good yield. The expected composition was confirmed by EDX analysis. It was shown that the complex interact with DNA through intercalative binding mode which was checked using UV-Visible Spectroscopy. The intercalative mode of binding was also confirmed through viscometry and Molecular docking studies. The result of the Molecular docking study revealed that complex interacts with one H-acceptor and two pi-Hs with DC3 and DG4 residues of DNA, respectively. The results of DNA damage activity show that the synthesized complexes compared to H 2 O 2 (control) have no obvious DNA laddering profile. Further the lower DNA damage activity of these complexes enhances their importance in drug development field. The hemolytic activity results exhibited that the screened complexes showed concentration dependent hemolysis. The POM analysis were also carried out which demonstrated that the synthesized complexes might be an innovative addition in drug development. The lack of DNA degradation ability and cytotoxicity to human blood erythrocytes increment the significance of these complexes in the field of drug development.