Synthesis and SAR Studies of Potent Antioxidant and Anti-Inflammatory Activities of Imidazole Derived Schiff Base Analogues

A novel sequence of imidazole derived Schiff base analogues 4-23 were synthesized and characterized by spectroscopic and analytical techniques. The in vitro antioxidant activities of these compounds were evaluated by using DPPH, ABTS and DMPD assay. The results exposed that the IC50 of compounds 9, 10, 11, 15, 16, 22 and 23 were lower than that of standards in all the three tested antioxidant assays indicating good activities of these compounds. In vitro anti-inflammatory activities of the synthesized compounds were tested and the outcomes of results were confirmed that the compounds 5, 6, 7, 8, 12, 13, 14 and 21 exhibited excellent anti-inflammatory activity. Preliminary structure-activity relationship revealed that the compounds 9, 10, 11, 15, 16, 22 and 23 with electron donating moiety (OH, OCH3) were found to be excellent antioxidants and compounds 5, 6, 7, 8, 12, 13, 14 and 21 with electron withdrawing moiety (Cl, F, NO2 and Br) were found to be excellent anti-inflammatory agents. Synthesis and SAR Studies of Potent Antioxidant and Anti-Inflammatory Activities of Imidazole Derived Schiff Base Analogues Shantharam CS1, Swaroopa M2, Darshini N3, Mallesha N3* and Rakesh KP3* 1Department of Chemistry, Pooja Bhagavath Memorial Mahajana Education Centre, Mysore-570016, Karnataka, India 2Department of Pharmaceutical Chemistry, Bharathi College of Pharmacy, Mandya-571422, Karnataka, India 3Sri Ram Chem, R & D Centre, Plot No 31, JCK Industrial Park, Belagola Industrial Estate, Mysore-570016, Karnataka, India


Introduction
Medicinal chemistry is an interdependenty established science that encompasses the innovation, progress, recognition and revelation of the mode of action of biologically dynamic compounds at the molecular level [1]. Heterocycles form by far the most of classical divisions of organic chemistry and are of vast use in biologically and industrially. Heterocyclic nucleus imparts an essential function in medicinal chemistry and serves as a key template for the improvement of various therapeutic agents [2]. Imidazoles have in use a sole arrangement in heterocyclic chemistry, and its derivatives have attracted significant interests in recent years for their multipurpose properties in chemistry and pharmacology. It improves pharmacokinetic characteristics of pilot analogues and thus is used as a remedy to optimize solubility and bioavailability parameters of projected unsuccessfully soluble lead molecules. The imidazole derivatives possess extensive spectrum of biological activities such as anti-inflammatory [3], anti-oxidant [4], anti-bacterial [5], anti-cancer [6], anti-tubercular [7] and anti-HIV [8] activities etc.

Literature Review
Antioxidant participate a fundamental role in the defense mechanism against oxidative damage induced by free radicals and reactive oxygen species (ROS). Reasonable ROS invention and detoxification in a common cellular metabolism is significant to keep the mammalian cells in healthy condition. When a cell fails to detoxify the excessive ROS generated as a result of destructive species or low level of antioxidants, they enter into a state of oxidative stress and are smashed [9]. High levels of ROS can cause injure to cell arrangement, nucleic acids, membrane lipids and proteins [10]. They in addition damage purine and pyrimidine bases of DNA molecule, thus most important to mutation [11]. Oxidative stress on a cell due to high level of ROS can lead to several disorders including cancer, neurodegenerative disorder, atherosclerosis and aging [12]. A lot of studies have recommended that antioxidants that can deactivate free radicals may be of essential interest in the preclusion of vascular diseases and some forms of cancer [13].
Non-steroidal anti-inflammatory drugs (NSAIDs) are commonly approved medications in the earth. They are mainly used for the treatment of pain, fever and inflammation, particularly arthritis [14]. Rheumatic diseases are the most common causes of disability in European countries and NSAIDs are still commonly used remedies. Chronic use might cause several serious adverse effects, the most important one being gastric injury and renal complications. Gastrointestinal (GI) injure from NSAIDs is usually recognized to two factors: local irritation by the straight contact of the free carboxylic acid (COOH) moiety of NSAIDs with GI mucosal cells and decreased tissue prostaglandin invention in tissues [15].
In view of above facts and in extension of our drug development series [16][17][18], the present work involves the synthesis of a new sequence of imidazole derived Schiff's bases as potential anti-inflammatory and antioxidants. A number of reported effective antioxidant and antiinflammatory activities of Schiff's bases are shown in Figure 1 [19][20][21].

Biology
Antioxidant activities: In vitro antioxidant activities of all the synthesized compounds were evaluated by DPPH [22], ABTS 23 and DMPD 24 cation radical scavenging assay. A lower IC 50 value indicated good antioxidant activity. The results were shown in Table 1. Some of the synthesized compounds showed strong antioxidant activities.   In outlook of the above points, compounds having -OH (phenolic) and -OCH 3 (anisole) groups in the phenyl ring (9, 10, 11, 15, 16, 22 and 23) were found to be the most potent antioxidants. In phenyl ring the number of hydroxy and methoxy group increases the activities also increases [16]. The compounds with electron withdrawing Cl, F, NO 2 and Br substituents (5-8, 12-14 and 21) showed least antioxidants activity and IC 50 values are higher than the standards. The compounds 17-20 were containing both electron donating and withdrawing groups shown that average activity.

General process
All necessary chemicals and reagents obtained from Merck (India) and Avra Synthesis (India) were used without further purification. Melting points were determined on a Thermonik melting point apparatus (Mumbai) and are uncorrected. FT-IR was performed using a Jasco spectrometer (Japan) using nujol media. 1 H NMR (400 MHz) and 13 C NMR (100 MHz) spectra were recorded on a Agilent Technologies (USA) using DMSO (d 6 ) as solvent. High resolution mass spectroscopic analysis was performed on a Bruker MicroTOF QII mass spectrometer in positive mode. Development of the reaction was monitored by aluminum coated TLC plates with the solvent system comprising chloroform/methanol in the ratio 98:02 (R f a ) and 95:05 (R f b ) and the compounds on the TLC plates were detected by under UV light.

5-(1-Hydroxy-1-methyl-ethyl)-2-propyl-3H-imidazole-4carboxylic acid hydrazide (3)
To a solution of 2 (10.5g, 0.043 mol) in ethanol (100 mL), hydrazine hydrate (2.6g, 0.052 mol,) was added. The reaction mixture was refluxed for 16 hrs for completion of the reaction (monitored by TLC). The solvent was removed under reduced pressure and cooled by adding ice cold water. The resulting precipitate was filtered, washed with cold water and recrystallized from ethanol to get the desired compounds 3.

-( 2 -
Percent inhibition was calculated from the following equation:

ABTS (2,2-azinobis-(3-ethylbenzothiazoline-6-sufonic acid) test:
The ability of the test sample to scavenge ABTS .+ radical cation was determined according to the literature method [23]. The ABTS .+ radical cation was pregenerated by mixing 7 mM ABTS .+ stock solution with 2.45 mM potassium persulfate and incubating for 12-16 hrs in the dark room at normal temperature till the colors changes from green to blue, then the absorbance was stable. Using distilled water, the absorbance of the ABTS .+ solution was equilibrated to 0.70 (± 0.02), then 2 mL was mixed with different concentration of the test sample (20 to 100 µg/ mL) and after 6-8 min the absorbance was measured at 734 nm.
The scavenging effect of ABTS .+ radical was calculated using the following formula:

ABTS .+ scavenging effect (%) = [(A c -A s ) /A c ] × 100
Where, A c is the initial concentration of the ABTS .+ and A s is the absorbance of the remaining concentration of ABTS .+ in the presence of compounds.

DMPD (N, N-dimethyl-p-phenylenediamine) test:
The DMPD radical scavenging ability of synthesized compounds was determined by the Fogliano et al., method [24] with slight modification. The 5 mL solution of DMPD (105mg) in distilled water was prepared. Then, 1 mL of this solution was added to 100 mL of 0.1 M acetate buffer (pH 5.3). 0.3 mL ferric chloride (0.05 M) was to the solution to produce DMPD .+ . Different concentrations of standard antioxidants or synthesized compounds (20-100 µg/mL) were added, and the total volume was adjusted to 1 mL with distilled water. 1 mL of the DMPD .+ solution was added to the reaction mixture. The reaction mixtures were incubated in the dark for 15 min. The absorbance was measured at 505 nm.

Anti-inflammatory activity
Human erythrocyte suspension: The human blood was collected from a healthy volunteer who had not taken any NSAIDs for 2 weeks prior to the experiment and collected in heparinzed vacutainer. The collected healthy human blood was washed 0.9% saline and centrifuged for 10 minutes at 3000 rpm. The packed cells were washed with 0.9% saline and 40% v/v suspension made by isotonic phosphate buffer of 154 mM NaCl in 10 mM Sodium Phosphate Buffer at pH 7.4 used as Stock erythrocyte or RBC suspension.

Hypotonic solution-induced haemolysis:
The activity of the synthesized compounds was performed according to the reported method. 25 The test sample consisted of stock erythrocyte (RBC) suspension 0.5 mL mixed with 5 mL of hypotonic solution (50 mM NaCl in 10 mM Sodium Phosphate Buffered saline at pH 7.4) containing different concentrations of sample (20, 40, 60, 80 and 100 µM/mL). The control consists of 0.5 mL RBC suspension mixed with 5 mL of hypotonic buffered solution alone. The mixtures were incubated for 10 minutes at room temperature, centrifuged for 10 minutes at 3000 rpm and supernatant was measured by spectrophotometrically at 540 nm. The % inhibition of haemolysis was calculated from the following formula. Where: A 1 = Absorbance of hypotonic buffered solution alone.
A 2 = Absorbance of test /standard sample in hypotonic solution.

Conclusion
In conclusion, we have synthesized a sequence of small and simple imidazole derived Schiff's base derivatives with various groups in benzene ring. All the synthesized analogues, compounds 9, 10, 11, 15, 16, 22 and 23 with OH and OCH 3 groups in benzene ring exhibited stronger radical scavenging activities than BHT and BHA in all the three assays performed. Compounds 5, 6, 7, 8, 12, 13, 14 and 21 with Cl, F, NO 2 and Br in benzene ring demonstrated excellent antiinflammatory activity than aspirin and indomethacin.