Multicomponent Synthesis of 2, 4, 6-Triarylpyridine Analogues As Antibacterial And Antiurease Agents

Five new 2,4,6-triarylpyridine analogues with potential biological importance have been synthesized using the multicomponent reaction (MCR) solid support catalyst synthetic approach. This green synthesis furnished method simplicity, reduce reaction time, and excellent yield, along with an additional feature of catalyst reusability. A number of spectroscopic techniques such as 1 H-NMR, IR, and mass spectrometry, conrmed structures of the newly synthesized compounds. Antibacterial and anti-urease activities of these compounds were evaluated. Results revealed that compounds 4a, 4c, 4d, and 4e exhibit signicant inhibition against gram-positive bacterial pathogens. Furthermore, all synthesized compounds showed potent urease inhibitory activity with IC 50 values ranging from 12.8 ± 1.04 to 23.7 ± 0.23 µM when compared with the standard inhibitor thiourea (IC 50 21.0 ± 0.23 µM). In


Introduction
Microbial and viral infections continue to be a major health problem and a great challenge worldwide due to their potential resistance. Medicinal chemists have been persistently struggling to develop novel and effective antibiotics to overcome this problem (Mouradzadegun, Elahi, Ghanbarzadeh, Silicon, & Elements, 2015). In this context, pyridine analogues have received considerable attention due to their unique structural features and diverse biological properties, which range from antibacterial, to antitumor (Ren & Cai, 2009). Examples of such compounds that are marketed pharmaceuticals include esomeprazole, used to treat symptoms of gastroesophageal re ux disease (GERD), and can lower the risk of bleeding after endoscopy in patients with ulcers. Other examples include rimiterol and etoricoxb (Cotton et al., 2000). Rimiterol is a direct-acting sympathomimetic with predominantly βadrenergic activity and a selective action on β2-receptors; it is a third-generation short-acting β agonist. Etoricoxb, on the other hand, is a selective inhibitor of cyclooxygenase-2 (COX-2), an enzyme involved in pain and in ammation, and is a nonsteroidal anti-in ammatory drugs (NSAIDs). Among other classes, 2,4,6-triarylpyridines are of great interest due to their wide range of biological and therapeutic potential.
Published research indicated that a large variety of catalysts such as heteropolyacid H 14   . However, these syntheses suffer from a number of shortcomings including the use of expensive catalysts, and the removal of catalyst and additional puri cation steps, thus compromising green chemistry principles. Another challenge is to construct multifunctional structures with linear approach that suffers from overall low yield. To overcome these problems, the one-pot MCR approach has been adopted to afford structural complexity and diversity in a single step which is cost effective (Sunderhaus & Martin, 2009). Accordingly, we have used the MCR method under solvent-free conditions, which is ecofriendly, and using a solid support silica impregnated with acid catalyst in the synthesis of new triarylpyridine analogues.
On the other hand, quantum chemical calculations are fascinating tools in chemistry as they rationalize the properties of newly synthesized compounds (A. J. C. m. s. Irfan, 2014). In this respect, the density functional theory (DFT) was comprehensively used to engross the geometric and electronic properties, which successfully reproduce experimental evidence (A. Irfan ). In the present study, optimizations were performed by B3LYP functional and triple zeta with 2 polarization function (TZ2P) basis sets. All calculations in this work were conducted using the Amsterdam Density Functional (ADF) modeling suite. Accordingly, we describe herein the synthesis and characterization of these new compounds (Schemes 1), along with the evaluation of their antibacterial and anti-urease activities. To the best of our knowledge, no previous study of this kind has appeared in the literature.

Materials and Methods
All chemicals used throughout this investigation were purchased from Sigma Aldrich, Fluka and Merck, and were used as received. Progress of reaction and purity of products were monitored by thin layer chromatography (TLC) carried out on packed on silica gel Al (aluminum) sheets (60 F254). Plates were visualized under UV light, where appropriate. Catalyst was characterized by means of a scanning electron microscope (SEM), with accelerating voltage 0.2-30 kV of 50/60 Hz power by (Carl Zeiss Company).

Catalyst preparation
Catalyst was prepared according to a published procedure which involved stirring a mixture of conc. sulfuric acid and silica gel in dichloromethane for 4 h; this has afforded a white amorphous powder, which was used as a solid support medium without further puri cation (Riego, Sedin, Zaldívar, Marziano, & Tortato, 1996).
General procedure for preparation of 4a-e Compounds 4a-e were prepared by the following procedure: Equimolar amounts of arylaldehydes (1ae), 2-acetylfuran (2), 2-acetylthiophene (3), NH 4 OAc (1.5 mole %), and an appropriate amount of silica catalyst were mixed in a round bottom ask and re uxed at 80 o C for 1 h ( Table 1). Progress of reaction was monitored by TLC using (10 %) a mixture of n-hexane and ethyl acetate as a solvent system. Upon completion of reaction, the mixture was allowed to cool to room temperature, dissolved in methanol, and ltered. The ltrate was poured onto crushed ice to afford the nal products (4a-e). Using the same general procedure, the following compounds were prepared: We evaluated the antibacterial activity of synthesized compounds 4a-e using the disc diffusion method. Brie y, test compounds (10 mg/mL) were applied on the pasteurized lter paper discs, dried overnight at room temperature, and stored; the negative control discs were prepared using the same procedure. In a standard microbiological working environment, bacterial cultures were grown overnight at 37 o C in nutrient broth medium and spread onto solidi ed nutrient agar medium in petri dishes. Then control and test discs were applied onto the solidi ed medium surface and incubated at 37 o C for 12-15 h. Results were calculated by evaluating the zone of inhibition in millimeters for each compound and compared with sparaxin, used as a standard (Pervez et al., 2008).

Urease Inhibition
Investigation of the urease inhibition activity of compounds 4a-e was carried according to the procedure outlined by Khan (2017) Fig. 3S and Fig. 4S). The Si-H 2 SO 4 catalyst was successfully employed for the synthesis of ve novels 2,4,6-triarylpyridine derivatives using various aryl-aldehydes (R 1 ), and results are shown in Table   1. The proposed mechanism for the construction of 2,4,6,-triarylpyridine ring involved aldol condensation of 2-acetyl furan enol (I) with 2-acetyl thiophene to furnish α,β-unsaturated ketone (III) catalyzed by Si-H 2 SO 4 . Then, activated thiophene enamine (V) was added to the double bond of (III) in a Micheal addition fashion to form (VI), which ultimately converts to its keto form (VII). This intermediate then undergoes hetroannulation by a nucleophilic attack of nitrogen on the carbonyl carbon to afford triaryl dihydropyridine (VIII), which upon oxidation yields the desired products ( Fig. 1)

Optimization of Reaction Conditions
In order to nd the optimum loading of catalyst, equimolar amounts of 2-acetyl furan, 2-acetyl thiophene, benzaldehyde, and NH 4 OAc (1.5 mole %) were mixed. In this regard, three parameters were explored for reaction optimization including catalyst ratio, catalyst amount, and temperature. Initially, several attempts were made with various ratios of Si-H 2 SO 4 catalyst ( Table 2). Results showed that the Si-H 2 SO 4 catalyst with 1: 0.5 ratio proves an e cient catalyst, which led to a single spot on TLC for 2,4,6-triarylpyridines synthesis with minimum time as compared to other ratios. Then, the effect of catalyst amount has been scrutinized, which revealed that with increased amount, reaction rate and product yields also increased. Results indicated that 1 g of the catalyst was su cient for the target compound. However, 1.2 g decreased the product formation, which could be due to increased acidity of reaction system. Finally, the effect of temperature was investigated by carrying out the same model reaction at different temperatures, where good t yield obtained at 80 o C (Table 3). Finally, reusability of the catalyst is one of the most signi cant bene ts of this strategy that makes the method cost effective. In this respect, the catalyst was ltered off, washed with water, dried, and reused for the same reaction. The catalyst could be recycled at least four times with only slight reduction in the catalyst activity.

Biological Activities
In Vitro Cytotoxicity The synthesized derivatives, 4a-e, were evaluated for cytotoxicity assay against 3T3 (mouse broblast) cell lines and exhibited IC 50 > 30, suggesting they are non-cytotoxic. Experimental results were compared with the standard drug cyclohexamide with an IC 50 value of 0.19 ± 0.11 µM.

Antibacterial Screening
We evaluated the antibacterial activity of compounds 4a-e against four Gram-negative (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Shigella exneri) and two Gram-positive (Bacillus subtilis, Staphylococcus aureus) bacterial strains. Results revealed that compounds 4a, 4c, 4d, and 4e exhibit signi cant antibacterial activity against Gram-positive pathogens as shown in Table 4. Urease Inhibition Activity Listed in Table 5 are the urease inhibitory activity of compounds 4a-e. Results reveal that compound 4a exhibits potent urease inhibition with an IC 50 value of 12.8 ± 1.04 µM when compared with the standard inhibitor, thiourea (IC 50 = 21.0 ± 0.23 µM). Results also show that compound 4a exhibits remarkable inhibition activity, and is more potent than the standard drug. This could be due to π-π stacking interactions of the phenyl group at C-4 of the pyridine ring. Inhibition activity of compound 4b is lower than that of 4a with an IC 50 of 19.6 ± 1.68 µM; this might be due to the presence of an electron-donating group (-OH ) at the ortho position of phenyl ring bonded to C-4. Similarly, compound 4c exerted signi can inhibition with an IC 50 of 15.8 ± 2.23 µM, due to the methoxy group at the para position of phenyl moiety attached to C-4 of the pyridine ring. On the other hand, the inhibition activity of compound 4d could be ascribed to the presence of hydrophilic interactions of furan and thiophene moieties. Furthermore, π-π stacking interactions of the substituents may lead to the signi cant activity of compound 4d, which has an IC 50 value of 17.9 ± 1.31 µM as compared to standard. Finally, compound 4e was the least active as urease inhibitor with an IC 50 of 23.7 ± 0.23 µM; this may be due to decreased ππ stacking interactions due to phenyl and chloro moieties.   Table 7. Data show that E HOMO-1 at S 0 decreases in the order: 4d (-6.22) > 4b (-6.27) = 4c (-6.27) > 4c (-6.37) > 4a (-6.45 Finally, the EDG -OCH 3 causes an increase in E gap while 4-furan and -Cl reduce its value as compared to compound 4a.  Stewart, 1979) [23,24] as well as by computational means. MEP illustrates the wide-ranging electronic and nuclear charge distribution, which is an appropriate feature to understand the reactivity of various species (Murray & Politzer, 2011). Depicted in Fig. 3 is the MEP mapping of compounds 4a-e, illustrated in color visualizations. The red color denotes higher negative potential regions, which are required for electrophilic attack, whereas the blue color identi es the higher positive potential regions favorable for nucleophilic attack. In this respect, the MEP decreases in the order blue > green > yellow > orange > red; the red color shows the strongest repulsion while blue elucidates su cient attraction.
In 4a, the negative electrostatic potential is located on the oxygen of furan and nitrogen of pyridine, whereas the positive electrostatic potential is found on the phenyl and thiophene moieties. In 4b, Similarly, Hirshfeld charge on C4 was -0.044 in 4a while substituting with the EDG -OCH 3 lead to changing the value to 0.080. The Hirshfeld charge on C3 was found -0.044 in 4a while by introducing EDG -OH lead to change its charge -0.073 in 4b. In addition, Hirshfeld charge on C3 was -0.044 in 4a while by substituting EDG -OCH 3 lead to change its value, i.e., --0.062. The Hirshfeld charge on C5 was found -0.044 in 4a while by introducing EDG -OH/OCH 3 lead to change its charge -0.060/-0.071 in 4b / 4c, which is due to the donating effect of -OH/OCH 3 . Replacing 4-phenyl by 4-furan moiety reduced the positive charge at C7 from 0.015 to 0.007 in 4c. The electron-withdrawing group (EWG) -Cl at the para position of the phenyl group reduced the negative charge at C2, C3, C4, C5 and C6 atoms. However, the most signi cant effect was observed at C4 where the negative charge -0.044 has become 0.018 due to the withdrawing effect of -Cl. The Hirshfeld charges revealed that -OH and -OCH 3 at the ortho and para positions increase the negative charges on C3 and C5 atoms, suggesting that more negative charge on these sites in 4b and 4c would be favorable for electrophilic substitution.

Conclusions
In summary, we have synthesized ve new 2,4,6-triarylpyridine derivatives in good yield A using the solid support catalyst approach. The present methodology offered attractive features such as short reaction time, simplicity, and reusability of the catalyst. The prepared compounds were tested in vitro for antibacterial and urease inhibition activities. Results revealed that all of these newly synthesized compounds display good to moderate antibacterial and urease inhibition potential, and may serve as lead molecule in the drug discovery of new antibacterial drugs. In addition, the electronic properties of these derivatives have been determined with various methods such as DFT and Hirshfeld charges method. The Table 6 is not available.

Scheme
Scheme 1 is available in the Supplementary Files section. Figure 1 Proposed Mechanism for a one-pot Synthesis of 2,4,6-triarylpyridines   Hirshfeld charges of compounds 4a-e

Supplementary Files
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