Cu(OAc)2 AS A GREEN PROMOTER FOR ONE-POT SYNTHESIS OF 2-AMINO-4,6- DIARYLPYRIDINE-3-CARBONITRILE AS ANTIBACTERIAL AGENTS

The extensive use of antimicrobial drugs and their resistance against bacterial infections have led to discover new antimicrobial compounds. In this study, we wish to report, one-pot synthesis of 2-amino-3cyanopyridine derivatives (1a-14a). These compounds were synthesized in the presence of Cu(OAc)2 as a highly effective heterogeneous acid catalyst. Here we evaluated the antimicrobial activities of these compounds against different species of microorganisms including gram positive and gram negative bacteria as well as fungi. Standard antimicrobial methods include disc diffusion and Broth microdilution method according to the protocol of the Clinical and Laboratory Standards Institute (CLSI). Synthesis of 2-amino-3-cyanopyridine derivatives were done via reaction of aromatic aldehydes, acetophenone derivatives, malononitrile and ammonium acetate in the presence of Cu(OAc)2 under reflux condition. The results show compound 2-amino-6-(4-chlorophenyl)-4phenylnicotinonitrile (10a) had the best antimicrobial efficacy toward C. albicans, E. faecalis, P. aeroginosa and E. coli. In conclusion, comparing the structure and activity of the compounds (10a), this compound with the presence of Cl residue at para-position of phenyl ring improves the antibacterial and antifungal activity.


Chemistry
All compounds were purchased from Merck and Fluka chemical company and used without any additional purification. FT-IR spectra were run on a Bruker, Eqinox 55 spectrometer. A Bruker (DRX-400 Avanes) NMR was used to record the 1 H-NMR spectra. Melting points were determined by a Buchi melting point B-540 B.V.CHI apparatus and were uncorrected. The products were characterized by FT-IR, 1 H-NMR, and their physical properties were compared with those reported in the literature.
Typical procedure for preparation of 2-amino-4,6-diarylpyridine-3-carbonitrile in the presence of Cu(OAc) 2 In a 25 mL round bottom flask, aldehyde (1 mmol), acetophenone derivative (1 mmol), malononitrile (1 mmol), ammonium acetate (1.5 mmol), Cu(OAc) 2 (0.1 g) and ethanol (3 mL) were charged and mixed under reflux condition. The progress of reaction was monitored by TLC. After completion of the reaction, the mixture was cooled and filtered to isolation of solid product. To obtain pure product, it was washed with ethanol.

Antimicrobial assays
Antimicrobial effect of all agents against two Gram-positive and two Gram-negative bacteria including Staphylococcus aureus (ATCC29737), Escherichia coli (ATCC15224), Pseudomonas aeruginosa (ATCC9027), Enterococcus faecalis and Candida albicans as fungus strain was carried out by two standard antimicrobial method include disc diffusion and Broth microdilution method according to the protocol of the Clinical and Laboratory Standards Institute (CLSI). A stock suspension of each synthesized agent was made in suitable solvent and final suspension was prepared in Mueller-Hinton Broth (MHB) media at 4500 to 1 µg/mL concentrations.

Primary disc diffusion method
Disc diffusion method was used for the primary evaluation of antimicrobial susceptibility of the tested agents. A total of 100 µL of each bacterial suspension (with turbidity of 1.5×10 8 CFU/mL) were streaked on brain heart infusion (BHI) agar plates separately. Then, sterile blank 6-mm filter paper discs contain 50 µL of each agent was placed on the plates. The plates were left under laminar air flow for 15 min and incubated for 24 h. After incubation, the zone of inhibition around each disc was measured. Paper disc containing solvent and sterile water were used as control group and negative control, respectively. Also, antibiotic standards for each microorganism according to performance standards for antimicrobial susceptibility testing were applied for standard control. These procedures were repeated for 6 times.

Determination of minimum inhibitory (MIC) and bactericidal (MBC) concentrations
To prepare bacterial suspensions for inoculation, microorganisms were cultured in MHB up to reach turbidity of the 0.5 McFarland standards (equivalent to OD600 = 0.12) using a biophotometer, then were diluted 1:20 to yield 5×10 6 CFU/mL. Using a 96-well micro plate containing 90 µL MHB medium with the desired concentration of each compound, 10 µL of prepared inoculums suspension was inoculated to obtain final microbial concentration at approximately 5×10 5 CFU/mL or 5×10 4 CFU/well. MHB without any agent and microbe was used as negative control, MHB cultured microorganisms were used as positive controls, ampicillin and gentamycin were applied as standard controls and blank wells contain MHB media with the desired concentration of agent with no bacteria inoculation. Optical density of each well was measured at OD600 by microplate reader (BioTek, Power Wave XS2) after 24 h incubation at 37 °C. In the case of fungi candida albicans, all microdilution tests were done in RPMI 1640 media.
MIC value was defined as the lowest concentration of antimicrobial agent in which 90% of the growth of microorganism is inhibited.
To determine MBC, three concentrations of each agent that was equal and higher than MIC, were spread on BHI agar plates and incubated for 24 h in 37 ºC incubator and then, the colonies were counted. The MBC was defined as the lowest concentration in which the number of colonies was less than 3 on each plate. These procedures were repeated for 3 times.

Statistical analysis
The results obtained from each antimicrobial test were analyzed using one-way analysis of variance (one-way ANOVA). Due to the presence of interaction effect, multiple comparisons were performed using One-way ANOVA/Tamhane post hoc tests to determine the differences between means. A P value of ≤0.01 was considered to be statistically significant. Values are expressed as means ± SD.

RESULTS AND DISCUSSION
Initially, we have carried out the four-component reaction of benzaldehyde (1 mmol), acetophenone (1 mmol), malononitrile (1 mmol) and ammonium acetate (1.5 mmol) in the presence of Cu(OAc) 2 under various conditions (Table 1). Ethanol was found to be the preferred solvent for this one-pot transformation under reflux condition in the presence of Cu(OAc) 2 (0.1 g) after 2 hours (Table 1, entry 7). At the beginning of the reaction, the substrates were dissolved completely in the medium to form a homogeneous mixture, but near the completion of the reaction, the system became a suspension, and the product precipitated at the end of the reaction. To our delight, almost full conversion of substrates into a product was found according to TLC. The mixture was allowed to cool at room temperature and then, the resultant precipitate was isolated by filtration, washed with cold ethanol carefully and recrystallized from ethanol. Scope of the synthesis of 2-amino-3-cyanopyridines was investigated in Table 2.
Various aromatic ketones are suitable substrates for the reaction. Electron-deficient aromatic aldehydes could react very readily at shorter time with higher yields. Aliphatic aldehydes gave just moderate yields. Surprisingly, while para-substituted and meta-substituted aromatic aldehydes tolerated very well for the transformation, ortho-substituted aromatic aldehydes gave trace products. There was also no report for the product coming from ortho-substituted aromatic aldehydes. Obviously, the reactivity of aldehyde is the key factor for this one-pot transformation. According to the above results, our proposed mechanism is depicted in Scheme 1.
In comparison to the compounds (10a) and (3a), based on variation of substitutions on the phenyl rings, we found that, the both compounds (10a) and (3a) have an Cl residue in the paraposition on the one of phenyl rings, but compound (10a) did not have any residue on the other phenyl ring, in the event that, compound (3a) have an methyl residue in para-position of the phenyl ring. Although compounds (10a) and (3a) showed the same antibacterial activities against C. albians.
As can be seen in Table 2, all compounds with nitro group in their structures did not show any antibacterial effect. Also compound (6a) with chlorine atom at meta-position of phenyl ring is not able to inhibit the antimicrobial strain. We can predict that for 2-amino-4,6diarylpyridine-3-carbonitrile compounds only chlorine atom at one para-position of the phenyl rings can be more effective than methyl or nitro groups at this position. Scheme 1. Suggested mechanism for the synthesis of 2-amino-4,6-diarylpyridine-3-carbonitrile derivatives.

CONCLUSIONS
In conclusion, an extremely efficient and green protocol has been developed for the synthesis of 2-amino-3-cyanopyridine derivatives via one-pot condensation of aldehydes, ketones, malononitrile, and ammonium acetate in ethanol in the presence of Cu(OAc) 2 as catalyst. This method tolerates most of the substrates with merits like ease of work-up, no side reaction, low costs and simplicity in process, and handling and environmentally benign catalyst. Biological studies showed that compound (10a) had good antibacterial effect on E. coli, P. aeruginosa, E. faecalis and C. albians strain. This compound with the presence of Cl residue in para-position of phenyl ring improves the antibacterial and antifungal activity.

ACKNOWLEDGMENTS
The Research Council of Yazd University and Shiraz University of Medical Sciences are gratefully acknowledged for financial support of this study. This project was supported financially from the Shiraz University of Medical Sciences by funding number 91-01-36-5251.