SYNTHESIS, CHARACTERIZATION AND BIOLOGICAL ACTIVITIES OF BIOINORGANIC COMPLEXES OF MOXIFLOXACIN.

Background: The growth of different organisms is controlled by drug-metal complexes which are injurious to humans. Moxifloxacin is one of the fourth generation fluoroquinolone antibiotics which inhibits DNA gyrase (a type II topoisomerase and topoisomerase IV). A number of studies were conducted on the Moxifloxacin-metal complexes regarding their biological applications. In this study synthesis and characterization of three moxifloxacin- biometal complexes with Zn(I), Ni(VI) and Co(VIII) was done and their antibacterial and antioxidant effects were studied. Methods: Moxifloxacin- zinc chloride, nickel chloride and cobalt chloride were synthesized by mixing solutions of zinc chloride, nickel chloride and cobalt chloride with the ethanolic solution of Moxifloxacin. These metal complexes were characterized by physio-chemical techniques such as We here report the synthesis and characterization of some novel and transition metal-moxifloxacin complexes including moxifloxacin–Zn(I), Ni(VI), and Cu(VII), [Zn(MOX) 2 ], [Ni(MOX) 2 ] and [Co(MOX) 2 ] . Characterization was done by FTIR, H1-NMR and UV-Vis. The antibacterial activity was determined by agar well diffusion method and antioxidant activities were performed by DPPH method.


Introduction:-
The rapid emergence of resistant bacteria is occurring worldwide, endangering the efficacy of antibiotics, which have transformed medicine and saved millions of lives. Many decades after the first patients were treated with antibiotics, bacterial infections have again become a threat. The antibiotic resistance crisis has been attributed to the overuse and misuse of these medications, as well as a lack of new drug development by the pharmaceutical industry due to reduced economic incentives and challenging regulatory requirements. (Blair et al., 2015;Ventola, 2015) In the past decades, only few new antibiotic classes (tigecycline from group of lipopeptides and tedizolid from group of oxazolidinones) have been developed and approved by FDA both of which provide coverage against Grampositive bacteria (Luepke et al., 2017) Gatifloxacin, moxifloxacin and Gemifloxacin became available for general use in 2004. (Andriole, 2005).
Moxifloxacin (MOX) is a fourth generation fluoroquinolone. It is chemically is [1cyclopropyl-7-(S,S)-2,8diazabicyclo(4.3.0)-non-8yl-6-fluoro-8-methoxy-1,4-dihydro-4-oxo-3quinoline carboxylic acid hydrochloride] (Kondaiah et al., 2017). Other Fluoroquinolones are classified into 4 generations nalidixic acid and cinoxacin (first generation), ofloxacin (OFX), norfloxacin, lomifloxacin and ciprofloxacin (CFX) (second generation), levofloxacin (LFX) (third generation) and gemifloxacin (GFX) (fourth generation). (Demir et al., 2017) Escalating resistance to moxifloxacin is being reported for many microorganisms, (Luepke et al., 2017;Murray et al., 2017) to overcome such resistance there are two ways, first to synthesis new antibiotics and second to prepare new drug delivery system. For synthesis of new antibiotic an average time required is ten years and at least 10 million dollars of money which is expensive and time consuming process (Fernandes & Martens, 2017).So generally the preparation of new drug delivery system of existing antibiotics is more appropriate to avoid the problem. The researchers have sufficient knowledge regarding the synthesis of moxifloxacin-metal complex. The biological activities and spectral studies of moxifloxacin is reported previously by W.F. El-Hawary et al. On other hand the synthesis, characterization and biological activities against E.coli, S. aureus, B. subtilis, Br.otitidis, P. aeruginosa of the moxifloxacin-metal complexes (Ti, Y, Pd and Ce) are reported in detail. (Sadeek et al., 2011). Studies on the synthesis, characterization and biological activities of moxifloxacin-imidazole-metal complexes is previously been done (Soayed et al., 2013). A number of researchers have studies on the synthesis, characterization and biological activities of the moxifloxacin-metal complexes. (Nakamoto & McCarthy, 1968;Soayed et al., 2013). However, the above discussed literature has not enough investigations and characterization of the moxifloxacin-metal complexes.
Moxifloxacin-metal complexes show good activity against both gram-negative and gram-positive bacteria. Fewer studies have been done with few metals complexed with moxifloxacin and very little biological activities are done on them. The synthesis of moxifloxacin-metal complexes with novel metals gave good result regarding their biological activities (Rafique et al., 2010) It is the inorganic chemistry which provides a good chance to use novel metal along with other transition metals to complex with drug and give greater biological activities.

Materials and Methods: -
Drug (Moxifloxacin) and metals (Zn, Ni, and Co) were purchased from the Sigma-Aldrich Limited. The reagents and chemicals were of analytical reagent grade and were used without further purification. All magnetic stir bars and glassware were washed with double distilled water.

Synthesis of drug-metal complexation
The Moxifloxacin (1 mMole) and metal ions (0.5 mMole) were dissolved separately in methanol. The solutions were mixed and refluxed for 8 -12 hours at 60 o C with continuous stirring. The resultant product was concentrated by evaporation and was precipitated using chilled chloroform. The precipitates were recrystalized.  then evenly swabbed onto labeled Mueller Hinton agar plates. The plates were allowed to dry for 5 minutes before punching wells with the help of sterile metallic borer (7 mm diameter). The dilutions of test compounds were introduced in wells and allowed to diffuse at room temperature before incubating at 37  C for 24 hours. Antibacterial activity was determined by measuring the diameter of growth inhibition zones (in mm). Growth inhibition was calculated with reference to the positive control i.e. moxifloxacin.   Table 1.

Antibacterial activity
Antibiotic resistance is one of the major problems in treating bacterial infections. The treatment of such resistant bacterial infections relies on the development of new compounds which are effective against a wide range of grampositive and gram-negative bacteria. In this study, therefore, various metal-moxicillin complexes were prepared and tested for antimicrobial activity.
The moxifloxacin alone showed moderate antibacterial activity for both gram-negative and gram-positive bacteria ( Table 2) compared to metal-moxifloxacin complexes which showed higher antibacterial activity (Table 3; Table 4; Table 5). As compared to the parent drug, moxifloxacin, the antibacterial activity of all tested metal complexes increased considerably. Among metal-moxifloxacin complexes, Ni(VI),) and Co(VIII) metal complexes showed higher antibacterial activity compared to Zn(I) metal complexes Table 6.
The Moxi-Zinc complex ( Table 3) showed highest activity against C. diphtheriae at the lowest concentration of complex used (MIC; 0.19 ppm). An antibacterial effect was also observed against S. typhi. (MIC; 0.38) compared to parent compound which showed no activity against it.
The Moxi-Ni complex showed highest antibacterial effect against S. aureus and S. epidermidis, (MIC; 0.19 ppm). Similarly, a higher antibacterial effect was also obtained against S. typhi (MIC; 1.55 ppm) Table 4.
The Moxi-Co complex were found to be most effective against E. aerogenes and C. diphtheriae (MIC; 0.19 ppm). Others including S. faecalis and K. pneumonia also showed higher sensitivity to this compound (MIC; 0.77 ppm) Table 5.
This study showed an overall high effectivity of metal-moxifloxicin componds against gram-negative and grampositive bacteria and could play a key role in new drug development. One of the major development in recent study is the effects of metal-moxi complexes against the Salmonella enterica serovar typhi, which are remarkably high (ranked as Moxi-Ni > Moxi-Co > Moxi-Zn). Emergence of drug resistant strains of S. typhi, classified as extensively drug resistance (XDR) typhoid, resistant against chloramphenicol, ampicillin, and trimethoprimsulfamethoxazole along with third-generation cephalosporins and fluoroquinolones are at rise (Klemm et al., 2018) with multiple cases reported in various parts of Pakistan (Ahmed, 2018). In this connection the discovery of highly effective metal-moxi complexes, against not only XDR S. typhi but also against other multidrug resistant organisms, is quite promising. While existing drug therapies are exhausting, these compounds could prove to be novel effective treatment options.

Antioxidant investigation:
Synthesis of new compounds having potential to protect the body from oxidative damage by neutralizing the free radicals, have become an important task in inorganic synthesis. Natural and synthetic antioxidants reduced the risk of chronic diseases by inhibiting the oxidation of substrate (Odeyemi et al., 2017). Various in vitro procedures provide a useful indication of antioxidant capabilities of compound by analyzing the capacity of compounds for radical capture or inhibition of radical formation. The in-vitro DPPH method is preferred over other methods because this method is convenient, reliable and fast. The DPPH free radical is scavenged by receiving hydrogen or 355 electron from antioxidant and become colorless in reduced form (Mishra et al., 2012). The percent inhibitions with relation to concentration of synthesized compounds along standard ascorbic acid have been reported in figure. Our analytical data showed ( Table 7) that with increase in concentration percent inhibition also increases, the maximum inhibition was found between 15-20 µg/ml (Biswas et al., 2010). Antioxidant activity influenced by the substitution at carbonyl group with metal groups, presence of different metal ions potentiates the scavenging effect. Synthesized compounds showed moderate to good antioxidant activates as compared with standard ascorbic acid (Table 7).

Conclusions: -
In the above research work, Zn(I), Ni(VI) and Co(VIII) moxifloxacin metal complexes were prepared and characterized by 1H-NMR, FTIR and UV-Vis. The IR spectra lead to the conclusion that in all complexes, ligand acts as a bidentate. These metal complexes have shown significant antibacterial effects against the tested bacteria. The moxifloxacin-Ni(VI), and Co(VIII) metal complexes showed high antibacterial activity compared to moxifloxacin-Zn(I) and moxifloxacin. The antibacterial activity indicates the metal complexes have more biological activity than parent drug (moxifloxacin). Antioxidant activity influenced by the substitution at carbonyl group with metal groups, presence of different metal ions potentiates the scavenging effect. Synthesized compounds showed moderate to good antioxidant activates as compared with standard ascorbic acid. Thus moxifloxacin metal compounds have potential to be novel antibacterial agents.