Potent Antibacterial Profile of 5-Oxo-Imidazolines in the New Millennium

Pharmaceutics and therapeutics industries enforced chemists to seek/discover antibacterial novel heterocycles owing specific bioactivity and innate characteristics significance. This chapter summarized potent antibacterial profile of 5-oxoimidazolines in the new millennium as an antibacterial against Gram-positive and Gram-negative bacteria viz. B. thuringiensis, S. aureus, E. coli, and E. aerogenes is presented in this chapter. 5-(H/Br benzofuran-2-yl)-1-phenyl 1H-pyrazole-3carbohydrazides are condensed with 4-(arylidene)-2 phenyloxazol-5(4H)-one in acetic acid at elevated temperature to yield product 5-(H/Br benzofuran-2-yl)-N(4-arylidene-5-oxo-2-phenyl-4,5-dihydroimidazol-1-yl)-1-phenyl-1H-pyrazole-3carboxamides. Different substrates like 4-(arylidene)-2-phenyloxazol-5(4H)-one allowed to react with benzaldehyde hippuric acid to yield 5-oxo-imidazolines/ 5-oxo-4,5-dihydroimidazole. All synthesized 5-oxo-imidazolines were characterized via elemental analysis and FT-IR, H-NMR and mass spectra techniques. All 5-oxo-imidazolines assayed in vitro for inherent antimicrobial activity at different concentration against stated bacterial strains and compared with standard chloramphenicol. 5-Oxo-imidazolines (3a and 3c) with 125 μg/mL concentration showed excellent antibacterial profile against Gram-positive bacteria, B. thuringiensis, while other derivatives at different concentrations showed moderate antibacterial activity against Gram-positive bacteria, S. aureus and B. thuringiensis. Gram-negative bacteria like E. coli and E. aerogenes are tested at higher concentration (1000, 500, and 125 μg/mL) and found good-to-moderate antibacterial activity. Tested products found non-active against E. aerogenes for 125, 61, and 31 μg/mL concentration also inactive at conc. 31 μg/mL against E. coli.


Introduction
Imidazole is a planer five-member ring with molecular formula C 3 N 2 H 4 , containing three carbon atoms and two nitrogen atoms in 1 and 3 skeletal positions as depicted in Figure 1. This is an aromatic heterocyclic ring that's classified as a diazole family owing non-adjacent nitrogens in its skeleton.
Biological importance of 5-oxo-imidazoline: Literature survey indicated that the synthetic drugs/molecules incorporated with 5-oxo-imidazoline found to owe assorted biological/clinical significance and wide range of pharmacological activities as mention below: Solankee et al. [17] synthesized some 5-imidazolinones (5) and evaluated as anticancer agent.
Kathrotiya et al. [19] and co-workers reported a series of some new quinoline based imidazole-5-one derivatives (7) and evaluated them as antibacterial and antifungal agent.
Desai et al. [20] also reported the synthesis of 5-oxo-imidazole amides derivatives including quinoline unit (8) and assessed their antibacterial and antifungal agent.
Mohammad and coworkers [21] have prepared some new imidazolinones and investigated their antimicrobial activities. Khan et al. [22] have also reported antibacterial and fungicidal activity of 5-oxo-imidazolines. Herbicidal activity of imidazolinone derivatives have been reported by Andreani et al. [23]. Moreover Zhou et al. [24] and Pai et al. [25] have reported anticancer active analogues of 5-oxo-imidazolines. Imidazolinone derivatives which possess antifungal activities have been reported by Shah et al. [26]. Some new 5-oxo-imidazolines as antimicrobial agents have been investigated by Patel et al. [27]. Rao [28] have prepared substituted imidazolone derivatives and reported their pharmaceutical use as inhibitors of p38 MAP Kinase and ERK-2 inhibitors. Xue et al. [29] have synthesized and evaluated imidazole-2-one derivatives as potential antitumor agents. Parekh and co-workers [30] have synthesized 5-oxo-imidazolines as novel bioactive compounds derived from benzimidazole. Kanjaria and co-workers [31] have described imidazolinones as potential antimicrobial agents. Joshi et al. [3] have synthesized imidazolinones as potent anticonvulsant agents. Acharya et al. [32] tested the imidazolinone (9) having quinolone nucleus for their antibacterial activity toward Gram-positive and Gram-negative bacteria and antifungal activity toward Aspergillus niger at a concentration of 40 μg, they found active against microorganism.
In view of potent antimicrobial and other pharmacological activities exhibited by 5-oxo-imidazolines, a variety of novel imidazolone analogs (3a-g) were synthesized by the condensation of different substituted oxazolines (2a-g) with hetero-aromatic amines (1a-b). All the synthesized compounds were screened for in vitro activities against a panel of Gram-positive and Gram-negative bacteria.

Materials and method
Melting points of all synthesized compounds were recorded in open capillary tube and are uncorrected. IR was recorded on a Shimadzu IR Spectrophotometer in KBr pellets. 1H-NMR recorded on a Bruker AM 400 model (400 MHz) using tetramethylsilane (TMS) as an internal reference and DMSO-d6 as solvent. Chemical shifts are given in parts per million (ppm). Positive-ion electrospray ionization (ESI) mass spectra were obtained with a Waters MicromassQ-TOF Micro, Mass Spectrophotometer. Elemental analysis was done on Vario EL III Elemental Analyzer, all compounds showed satisfactory elemental analysis. Reactions were monitored by E. Merck TLC aluminum sheet silica gel 60F254 and seen spot in UV light and iodine chamber.

Experimental
(I) Synthesis of benzoyl glycine [33]: A solution of glycine (0.33 mol) in 10% NaOH (250 mL) of was prepared and benzoyl chloride (45 mL, 0.385 mol) was added to the above solution in portions. The mixture was shaken vigorously after each addition until all the chlorides have been reacted. The mixture was cooled by adding few grams of crushed ice and was acidified by adding conc. HCl slowly with constant stirring. The resulting crystalline precipitate of benzoyl glycine was filtered and washed with cold water and dried. The solid was treated with hot CCl 4 in order to remove benzoic acid. The dried product was recrystallized with boiling water.

(II) Synthesis of 4-(arylidene)-2-phenyloxazol-5(4H)-ones [33] (2a-g):
Benzoyl glycine (0.0476 mmol), aryl aldehydes (0.0476 mol), acetic anhydride (14 mL, 0.146 mmol) and anhydrous sodium acetate (0.0476 mmol) were placed in a 250 mL conical flask. It was heated on electric hot plate with constant shaking until the mixture liquefies completely. Then it was refluxed for 2 h on water bath. Then ethanol (10 mL) was added and mixture was allowed to stand overnight. The crystalline precipitate was filtered, washed with ice-cold alcohol and boiling water. The product was dried and recrystallized using benzene.

Common examination of the product
The newly synthesized compounds are soluble in following solvents which are listed in table also identification of newly synthesized compounds has been further confirmed by Lassaigne's test for nitrogen, all compound gives positive test. Table 1 represents the structure of all derivatives along with solubility solvent and Lassaigne's test.
IR spectrum of this 3a showed absorption bands at 3197 cm À1 due to ─NH stretching, disappearance of absorption band due to ─NH 2 stretching and two absorption bands at 1719 and 1640 cm À1 for two carbonyl groups of imidazoline and aryl amide respectively indicated that 4-(arylidene)-2-phenyloxazol-5(4H)-ones has condensed with 5-(5-H/Br benzofuran-2-yl)-1-phenyl-1H-pyrazole-3carbohydrazides to form 3a. In addition, 1 H NMR spectrum of 3a showed singlet at δ 10.65 ppm for ─NH group and disappearance of signal due to ─NH 2 group in the synthesized compound 3a which is expected in carbohydrazide 1a and also exhibited multiplet at δ 7.22-8.37 ppm due to 21 aromatic protons is in consistent with aromatic protons of 3a. The % of elements in 3a was C 74. 16 Similarly other imidazolinones (3b-g) were also identified on the basis of chemical transformation reaction, physical data, IR and elemental detection. IR spectra of each compound showed characteristics absorption bands for ─NH stretching and disappearance of absorption band due to ─NH 2 stretching, also showed corresponding band for carbonyl group. Elemental analysis was carried for nitrogen and sulfur of all compounds is found to be in good agreement with the calculated values.

Antimicrobial activity/profile
Antimicrobial activity means activity of any agent or drug against microbial organism. Microbial organism includes bacteria, viruses, fungi and protozoa. On the basis of their activity against specific microbial organism they termed as like antibacterial (against bacteria) that means they are capable to inhibit the growth of bacteria or to kill the bacteria. Other term is antifungal (against fungi), antiviral (against virus), antiprotozoal (against protozoa). Heterocyclic entities possess different antimicrobial activity. Activity changes by changing structural unit. It is very interesting thing to check out antimicrobial activity of newly synthesized compound. We carried out antibacterial activity of the novel compound (Figure 4).

Potent antibacterial/inhibition profile of 5-oxo-imidazolines (at different concentration) by agar disc-diffusion method
Test solutions were prepared with known weight of compound in DMSO and half diluted suitably to give the resultant concentration of 31-1000 μg/mL [35]. Whatman No. 1 sterile filter paper discs (6 mm) were impregnated with solution and allowed to dry at room temperature. In-vitro antibacterial activity was determined by using Mueller Hinton Agar obtained from Himedia Ltd., Mumbai. Petri plates were prepared by pouring 10 mL of Mueller Hinton Agar for bacteria containing microbial culture was allowed to solidify. The discs were then applied and the plates were incubated at 37°C for 24 h (bacteria) and the inhibition zone was measured as diameter in four directions and expressed as mean. The results were compared using chloramphenicol as a standard antibacterial agent. The results of antibacterial activity (i.e. zone of inhibition in mm) are given in the Tables 2 and 3.

Inhibition profile zone for Gram-positive bacterial strains of tested compound-3a-g
The synthesized compounds 3a-g were screened for their in vitro antimicrobial activity using agar disc-diffusion method against two Gram-positive bacterial   strains such as B. thuringiensis, S. aureus. Chloramphenicol was used as standard drug for bacteria. According to antibacterial data obtained the test compounds 3a-c at 125 μg/mL conc. showed excellent activity i.e. equal or higher than the standard drug and other derivatives viz. 3d-g at 125 μg/mL conc. showed good inhibitory activity against B. thuringiensis. At conc. 1000, 500, and 250 μg/mL imidazolinone derivatives 3a-g showed good to moderate activity against B. thuringiensis, whereas 3d and 3e are found to be inactive at 31 μg/mL against Gram-positive bacteria, B. thuringiensis. In case of S. aureus 3a exhibit with excellent activity at 63 μg/mL conc. While at 1000, 500, 250 μg/mL concentrations 3a-g possesses good to moderate activity. Whereas 3b & 3f are found to be inactive at 31 μg/mL also 3f found to be inactive at 125 μg/mL against S. aureus. Obtained results of in-vitro antimicrobial activities of 3a-h are summarized in Table 2.

Inhibition profile zone for Gram-negative bacterial strains of tested compound-3a-g
The synthesized compounds 3a-g were screened for their in vitro antimicrobial activity using agar disc-diffusion method against two Gram-negative bacterial strains such as E. coli, E. aerogenes. Chloramphenicol was used as standard drug for bacteria. According to antibacterial data obtained the test compounds 3a-g possesses good to moderate activity at higher concentrations, i.e. 1000, 500, 250 and 125 μg/mL against Gram-negative bacteria E. coli. At conc. 63 μg/mL 3a-g showed good activity while 3b & 3e found to be inactive against E. coli. At conc. 31 μg/mL 3d, 3f & 3g showed moderate activity whereas 3a, 3b, 3c & 3e found to be inactive against E. coli. In case of E. aerogenes, tested compounds showed moderate activity at higher concentrations while poor activity at lower concentrations. At conc. 125 μg/mL 3a, 3d, 3e and at 63 μg/mL 3a, 3b, 3d, 3e, 3f found to be inactive. All the compounds were inactive at a concentration of 31 μg/mL against E. aerogenes. Obtained results of in-vitro antimicrobial activities of synthesized 5-oxoimidazolines (3a-g) are summarized in Table 3. Standard drug: chloramphenicol.

Zone of inhibition (mm)
Bold value indicates activity of tested compound is equal or high than standard drug. Table 3.

Mechanism of inhibition/prohibition
Gram-negative bacteria habitually owe low susceptibility as outer membrane of their cell wall not gets blocked/penetrated by drugs easily and factors like amount of peptidoglycan, receptors, and lipids availability, nature of cross-linking, autolytic enzymes activity greatly influence the bio-activity, permeation, and incorporation of the antibacterial drugs. 5-Oxo-imidazoles showed their specificity for polysaccharides, thats present in the outer membrane of many Gram-negative bacteria and so acted selectively toxic for series of B. thuringiensis, S. aureus bacteria. Mechanistically, once alliance with lipopolysaccharide substrate in outer membrane of B. thuringiensis, S. aureus bacteria, synthesized imidazolinone: potent antibacterial agent changes their membrane structure, thus enhances permeability and disruption of osmotic balance that ultimately results higher physiological effects. Also, alteration like discharge of molecules from interior of B. thuringiensis, S. aureus bacterial cell inhibits respiration and increased water uptake may leads to cell death. Gram-positive bacteria own too thick cell wall and deny easy access of 5-oxoimidazoles via their bacterial cell membrane, thus less effective on E. coli and E. aerogenes series of bacteria. The inhibition profile zone for four different bacterial strains of tested compound-3a (at different concentration) 5-oxo-imidazoline compounds are shown in Figure 5.

Conclusion
Assorted antibacterial agents own certain limitations viz.; resistance/potency, vast types and numbers own different structures besides slightly dissimilar pattern of activity which made it necessary to discover/explore the existing class and functions of almost all the antibacterial agents. Thus, futuristic pathogenic bacterial infections/diseases can be easily cured via promising antibacterial chemotherapeutic agents derive from 5-oxo-Imidazole skeleton. Pursued chapter described antibacterial resistance of 5-oxo-imidazoles mostly against Gram-positive series like B. thuringiensis, S. aureus. 5-Oxo-imidazoles can act onto simple one-celled bacterial organism that could kill, inhibit, or at least slower down their growth and ultimately can inhibit concern diseases/infections. This chapter focused on helping futuristic researchers, clinicians, and academicians involved in synthesizing and corresponding biological screening of innate activity of certain novel imidazolinone heterocycles. These synthesized 5-oxo-imidazoles restrain potent antibacterial activity may own prospective different therapeutic behavior if developed as advanced drug moiety. Therefore, chapter focus on the basis of chemical structure of 5-oxo-imidazoles. Gram-positive and Gram-negative bacteria showed varied response/susceptibility toward 5-oxo-imidazoles.
Targeted 5-oxo-imidazolines (3a-g) a class of imidazolinones are successfully synthesized in good yields and purity checked by physical, analytical and spectral data. Antibacterial screening of 5-oxo-imidazolines (3a-g) exhibited a potent bactericidal. Thus, 5-oxo-imidazolines could be powerfully stimulates major advances in remarkable significant chemotherapeutics in medicine, biology and pharmacy. Overall these imidazolinones disturb macromolecules like cytoplasmic membrane covering cytoplasm which acts selective barrier to control internal composition of cell. 5-Oxo-imidazoles in particular interrupted such functional roles of cytoplasmic membrane and ionic outflow that resulted cell destruction/death. Synthesized potent bioactive 5-oxo-imidazoles may open new possibilities in the successful treatment of several diseases due to promising antibacterial profile. So, ample scope exists in further research of imidazolinones especially innate selectivity of 5-oxoimidazoles needs to carry out their chemotherapy as potent antibacterial aims to target cell membrane of range of Gram-negative bacteria as to derive novel drugs of new millennium.