SYNTHESIS AND EFFICIENCY OF NEW PYRIDINE, CHROMENE AND THIAZOLE CONTAINING COMPOUNDS AS ANTIMICROBIAL AND ANTIOXIDANT AGENTS

The versatile scaffold, N'-(2-cyanoacetyl)-2-hydroxybenzohydrazide (3) was utilized in the production of new pyridine, chromene and thiazole derivatives as antimicrobial and antioxidant agents. The synthetic strategy involves the treatment of precursor 3 with various arylidene-malononitrile and 3-aryl-2cyanoacrylate compounds to furnish substituted pyridines 5 and 7. The interaction of 3 with salicylaldehyde and/or phenyl isothiocyanate followed by cyclization with chloroacetone produced the corresponding 2-imino-2Hchromene-3-carbohydrazide and (thiazol-2-ylidene-acetyl)-salicylic acid hydrazide compounds 8 and 9, respectively. The structural features of the synthesized compounds were confirmed by using spectroscopic methods such as (IR, H NMR, C NMR and MS). The new pyridine, chromene and thiazole products showed potent antioxidants and antimicrobial activities. The thiazole derivative 9 exhibited the highest anti-bacterial and antifungal activities against S. aureus (75.0%) and B. subtilis (73.9%) and C. albicans (66.6%). The combination between salicylic acid hydrazide and thiazole moieties in the hybrid 9 indicated the best antioxidant activity (87.9%).


INTRODUCTION
The modern chemical industry is interested in producing nitrogen-, oxygen-and/or sulfurcontaining heterocyclic molecules due to their specific biological and pharmacological activities. Pyridine is the principal component in enormous natural and synthetic compounds of pharmaceutical interest [1,2]. Therefore, the interest in the preparation and application pyridinecontaining compounds is still desirable and continuous [3,4]. Many pyridine compounds have been described and characterized by their miscellaneous biological activities, such as anti-tumor [5], antituberculosis [6], antibacterial [7], and antihepatitis [8]. The synthesis of pyridine ring system relies upon one of two approaches: the condensation of carbonyl compounds [9][10][11] or cycloaddition reactions [12][13][14]. There are exceptions, such as ring expansion from 5-membered rings, but these approaches are generally low-yielding, narrow in applicability, or both. Also, thiazoles are one of the most active heterocyclic compounds which is characterized by a wide range of activities, such as antibacterial [15], antifungal [16], anti-inflammatory [17], antidiabetic [18], antioxidant [19], anticancer [20], antiviral [21][22][23], and antiprotozoal activities [24]. There are numerous protocols for construction of the thiazole ring system [25], the most broadly used synthetic routes are Hantzsch's Synthesis (the reaction of α-haloketone or aldehyde with thioamide), Gabriel Synthesis (the reaction of α-aminonitriles with dithioacid or esters, carbon disulfide, carbonyl sulfide, and isothiocyanates separately), and Tcherniac's Synthesis (αthiocyanatoketone on aqueous acid (HCl) or aqueous alkali-induced cyclization afforded 2hydroxy-4-aryl/alkylthiazole, while cyclization in dry ethereal HCl produced 2-chloro-4aryl/alkylthiazole). Chromene compounds are one of the most demanded compounds due to their unique pharmacological activity [26,27]. Chromene scaffolds display tremendous medicinal properties such as antimicrobial [28][29][30], antioxidant [31], antileishmanial [32], vascular- The synthetic plan of this work is based on utilization of the highly versatile N'-(2cyanoacetyl)-2-hydroxybenzohydrazide (3) in the building of new heterocyclic ring systems such as pyridine, thiazole, and/or chromene. Thus, the reaction of N'-(2-cyanoacetyl)-2hydroxybenzohydrazide (3) with several arylidene-malononitrile derivatives 4 proceeded by heating in ethanol and piperidine to furnish the corresponding N-(6-amino-4-aryl-3,5-dicyano-2-oxopyridin-1(2H)-yl)-2-hydroxybenzamides 5a-e (Scheme 2). The formation of pyridine ring system was started by Michael-type addition to form the intermediate A, which undergoes cyclization via intramolecular addition of N-H on the nitrile group. Air oxidation (loss of hydrogen molecule) of the produced intermediate B afforded the target pyridinone derivatives 5a-c. The structures of the produced pyridinone compounds 5a-c were elucidated by their spectroscopic analyses. The IR spectrum of pyridone 5c displayed the expected absorptions of the nitrile and carbonyl groups at 2209 and 1659 cm -1 , respectively. The 1 H NMR spectrum of pyridone 5c exhibited singlet at δ 3.84 ppm (-OCH3), multiplet and doublet signals (δ 7.14-8.10 ppm) for the aromatic protons, and two singlet signals at δ 8.20 and 11.28 ppm for the protons of N-H and O-H groups. The 13 C NMR spectrum of compound 5e displayed 18 signals for twenty carbon atoms. The carbon signals for the carbonyl groups resonate at δ 157.06 and 161.08 ppm.

Antimicrobial activity
In  18.5% against C. albicans. While pyridine compounds 5b and 7a (substituted with methyl group at the phenyl group) had no effect on both antimicrobial (anti-bacterial and anti-fungal). The MIC (minimum inhibitory concentration) for the potent pyridine, chromene and thiazole compounds 7c, 8, and 9 was assessed and the results are presented in Table 2.

Antioxidant activity
The newly synthesized pyridine, chromene and thiazole compounds were tested for their antioxidant activity by using both (DPPH and ABTS Assay). The results (Table 3) for ABTS assay indicated that: the thiazole compound 9 give the best antioxidant property (87.9%). The increasing in antioxidant activity of compound 9 may be because of the combination between salicylic acid hydrazide and thiazole moieties. For IC50 values of the synthesized products in relation to DPPH inhibition. The concentration of antioxidant obligatory to decrease the initial DPPH concentration by 50% (IC50) is the parameter that applied to indicate the antioxidant activity. According to the ABTS method, the IC50values of compounds 7c, 8 and 9 were 89.14, 102.63 and 33.27, respectively. The quantities of the ABTS test showed marginally lower scavenging capacity in relation to the DPPH method (Table 4) while IC50 values in the DPPH Method were 50.46 (compound 7c), 63.35 (compound 8) and 19.58 (compound 9). The tough antioxidant commotion of the synthesized products is record often connected with great content of total phenols. The extant effects determined that the antioxidant activity of thiazole compound 9 is very close to the standard antioxidant (ascorbic acid).

Screening of antibacterial and antifungal activity
The effect of antibacterial can well-known by testing the amalgamated pyridine, chromene, and thiazole compounds with four types of bacteria (E. coli, P. aeruginosa, S. aureus, and B. subtilis) and one type of fungi (C. albicans) by using the Agar Plate diffusion method. The investigated bacteria and fungi were grown in Muller-Hinton (MH) Media at 37 °C for 24 hours before being injected with 20L of 1 106 cfu/mL of the investigated microorganism in melted MH Media at 50 °C. Well was emptied into 9 cm diameter plates and left to solidify. The studied product was softened in DMSO to get a concentration of 5 mg/mL, and 100 mL was added to the conforming wells. The paper discs were saturated with the expected concentration of the complex solution before being placed in petri dishes containing nutritional agar media (agar 20 g + yeast extract 3 g + peptone 5 g), and each one was replicated three times [42,43].
Determination the minimum inhibitory concentration (MIC). In vitro, for antibacterial efficiency to examined the blended Product as reported by CLSI, 2015 and also in vitro for antifungal activity according to (CLS I M27 -A3 and CLS M38 -A2 Methods) (CLSI 2008). Two fold for the dynamic compound, make a serial dilution by weighted of 0.01 mL MH Broth for antibacterial and yeast peptone dextrose for antifungal assay [42]. For every filter paper which cut as circle and can be consider as well in occulated with 0.01 of the diluted bacterial suspension (5 x 10 6 cfu/mL) [43]. Ampicillin's antibacterial activity was calculated and utilized as a positive control [44].

Antioxidant assay
ABTS (5 mL of 7 mm) was mixed with 88 mL of 140 mm potassium persulfate to make the ABTS reagent. To measure the scavenging activity, 100 mL of ABTS reagent was combined with 100 mL of sample in a 96-well microplate and incubated at 25 °C for 6 m before being diluted with water (1:44 v/v). After incubation, the absorbance was measured at 734 nm using an Elisa reader. A 100 percent methanol control was also used as a control. Extrapolation from regression analysis yielded the IC50 ABTS values. The DPPH assay was performed three times, and the average values were used. A freshly prepared methyl alcohol solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical (0.004 percent w/v) was prepared and stored in the dark at 10°C.The test chemical was made into a methanol solution. A volume of 3 mL of DPPH solution was added to a 40 mL aliquot of the methanol solution. A UV-visible spectrophotometer was used to take instantaneous absorbance values. The decline in absorbance at 515 nm was monitored constantly, with data collected at 1 min intervals until the absorbance levelled off (16 min). All of the tests were repeated three times, and the results were averaged. The DPPH radical's percentage inhibition (PI) was estimated using the formula: PI = [{(AC-AT)/ AC} x 100]. Where AC = control absorbance at t = 0 min and AT = sample absorbance + DPPH at t = 16 min [45].

CONCLUSION
A series of new pyridine, chromene and thiazole compounds was synthesized through the reactions of N'-(2-cyanoacetyl)-2-hydroxybenzohydrazide with various arylidene-malononitriles, 3-aryl-2-cyanoacrylates salicylaldehyde and (phenyl isothiocyanate / chloroacetone). The structures of these synthesized compounds were confirmed by multiple spectroscopy techniques and screened for antimicrobial and antioxidant activity. The thiazole compound9 was the most potent of the tested compounds and showed the highest anti-bacterial and antifungal activities against S. aureus (75.0%) and B. subtilis (73.9%) and C. albicans (66.6%). The combination between salicylic acid hydrazide and thiazole moieties in the hybrid 9 indicated the best antioxidant activity (87.9%).