Synthesis and antimicrobial screening 6-Aryl-2-(Amino/methoxy)-4-[(4′-difluoro methoxy) (3′-hydroxy) phenyl] nicotinonitrile

A series of an easy and efficient route for the synthesis of some new 2-amino-3-cyanopyridine (4a-4i) and 2-methoxy-3-cyanopyridine (5a-5i) derivatives were synthesized through the reaction of various chalcone (3a-3i) with malononitrile in presence of ammonium acetate and sodium methoxide, respectively. The constitutions of the all synthesized compounds have been established by the IR, 1H-NMR, Mass spectral data and Elemental analysis. Furthermore, all the synthesized products were screened for their antimicrobial activity.


INRODUCTION
Pyridine functionalities have been widely studied [1,2] and widely used [3][4][5][6] but still generate much interest due to their wide range of application in medicinal chemistry [7][8][9][10][11]. The naturally occurring B6-vitamins pyridoxine, pyrodoxal, pyridoxamine and codecarbaxylase contain a pyridine nucleus [12]. Pyridine derivatives have been used as herbicides [13], for enrichment of cereals [14], for regulation of arterial pressure [15] and cholesterol levels in blood [16]. Some pyridines constitute an important class of antitumor compounds [17][18]. They also show antibacterial [19], antifungal [20], antimyotic [21] and antidepressant [22] activities. Among them, 3-cyanopyridines have been identified as IKK-β inhibitors [23]. Most derivatives are prepared by manipulation of pyridine and its simple homologues in a manner similar to chemistry of the benzenoid chemistry. However the simple pyridine compounds are prepared by the cyclization of aliphatic raw materials. The pyridine nucleus is found in a large number of commonly used drugs which have diverse pharmacological activities. Interests in the synthesis of multicyclic pyridine containing compounds have increased in recent years because of their biological and pharmacological activities.
The structure of synthesized compounds were assigned based on Elemental analysis, IR 1H-NMR and Mass spectral data. The antimicrobial activity was assayed by using the MIC (Minimum Inhibitory Concentration) method. All the compounds were screened in vitro for their antimicrobial activities against Gram +ve bacteria, Gram -ve bacteria and fungi. The biological activities of the synthesized compounds were compared with known standard drugs.

RESULTS AND DISCUSSION
The synthetic route adopted to obtain the 3-cyanopyridine derivatives 4a-4i and 5a-5i is shown in Scheme 1. 2-Amino-3-cyanopyridine derivatives 4a-4i were prepared from Chalcones 3a-3i by refluxing with malononitrile and ammonium acetate in Ethanol. The isolated product was crystallized with diethyl ether to get 2-amino-3-cyanopyridine derivatives in 58-83% yield. The 2methoy-3-cyanopyridine derivatives 5a-5i were prepared from Chalcones 3a-3i by refluxing with malononitrile and sodium methoxide in ethanol for 8 hrs. The isolated products were crystallized with diethyl ether to get 2-methoy-3-cyanopyridine in 54-83% yield. The structures of all newly synthesized compounds were assigned on the basis of spectral data such as IR, 1  The structural assignment of the title compounds 4a-i and 5a-i have been made on the basis of 1 H-NMR, Mass, elemental analysis and IR spectral studies which were in full agreement with the proposed structures. IR spectrum of compound 4e reveals absorption band in the region 3322 cm -1 corresponding to amine (C-N) stretching and 2218 cm -1 due to -CN. In 1 H-NMR spectra of 4e, the -CHF 2 protons absorbed as a singlet at δ 7.01 and broad singlet at δ 5.98 due to amine for 2H proton and rest of the aromatic proton appear at their respective position. Mass spectrum of 2-amino-6-(4′′chlorophenyl)-4-[(4′-difluoromethoxy) (3′-hydroxy) phenyl] nicotinonitrile showed (M+) peak at 387.8 which support the formation of product. The structure of 5a is interpreted from spectroscopic data. The IR spectrum of 5e showed a characteristic absorption band at 2218 cm -1 due to -CN stretching and 1546 cm-1 due to vinyl (C=C) stretching. 1 H-NMR spectrum of 5g reveals the presence of the -CHF 2 protons absorbed as a singlet at δ 6.93 and methoxy group at δ 3.82. Mass spectrum of 6-(4′′-methoxy phenyl)-4-[(4′-difluoro methoxy) (3′-hydroxy) phenyl] -2-methoxy nicotinonitrile showed (M+) peak at 447.2.

EXPERIMENTAL
All the melting points were determined on electro-thermal apparatus using open glass capillaries and are uncorrected. Formation of the compounds was routinely checked by TLC on silica gel-G plates of 0.5mm thickness, and spots were located by iodine and UV (254nm). The IR spectra were recorded on a Shimadzu FT-IR-8400 instrument using KBr pellet method. Mass spectra were recorded on Shimadzu GCMS-QP2010 model using Direct Injection Probe technique. 1 H-NMR was determined in CDCl3/DMSO-d6 solution on a Bruker AC 400MHz spectrometer using TMS as internal standard and coupling constants (J) are expressed in Hertz (Hz). Elemental analysis of the all the synthesized compounds were carried out on Elementar Vario EL III Carlo Erba 1108 model, and the results are in agreements with the structures assigned. All the reagents were purchased from Rankem (New Delhi, India) and Sigma-Aldrich (New Delhi, India) and are used without further purification.

ANTIMICROBIAL ACTIVITY
The newly synthesized compounds were screened for their antibacterial activity against Gram-positive (S. aureus ATCC 6538, M. luteus ATCC 9345), Gram negative (E. coli ATCC 4230, S. thyphi ATCC 14028) bacteria, as described by the guidelines in NCCLS-approved standard document M7-A4, using the micro dilution broth procedure [24]. Ampicillin trihydrate was used as the reference antibacterial agent. The antifungal activities of the newly synthesized chemical compounds were tested against yeast strain (C. albicans ATCC 14053) according to the guidelines in NCCLS-approved standard document M27-A2, using the micro dilution broth procedure [25]. Fluconazole was used as the reference antifungal agent. The solutions of test compounds and reference drug were prepared by dissolving in DMSO at a concentration of 2560 μg/mL. The 2-fold dilutions of the compounds and the reference drug were prepared (1280, 640, 320, 160, 80, 40, 20, 10 μg/mL). Antibacterial activities of the newly synthesized chemical compounds were performed in Mueller-Hinton broth medium at a pH of 7.2 with an inoculum of (1-2) × 10 3 cells/mL by the spectrophotometric method, and an aliquot of 100μL solution was added to each tube of serial dilution. The chemical compounds-broth medium serial tube dilutions inoculated with each bacterium were incubated on a rotary shaker at 37°C for 18 hr at 150 rpm. The minimum inhibitory concentration (MIC) of each chemical compound was recorded as the lowest concentration of each chemical compound in the tubes with no growth (i.e., no turbidity) of inoculated bacteria. Minimum inhibitory concentration (MIC, μg/mL) was measured and compared with control; the MIC values of the compound screened are given in below table no. II. From the result of biological evaluation, it has been observed that the compounds exhibited interesting biological activity, however with a degree of variation. Most of the compounds tested were found to have comparable antibacterial and exhibit low antifungal activity. From the table no. II, it can be observed that compounds 4a, 4c, 4d, 5a, 5b and 5i were moderate active against S. aureus, M. luteus, Escherichia coli , S. thyphi., C. albicans, Compounds 4g, 4h, 5d, 5e and 5h were give promising activity against S. aureus A, S. aureus, M. luteus, Escherichia coli , S. thyphi., C. albicans.

CONCLUSION
In summary, we have synthesized a series of novel 3-Cyanopyridines derivatives. All the newly synthesized compounds were confirmed with spectroscopic data like 1 H-NMR, Mass, IR Spectra, elemental analysis and evaluated antibacterial and antifungal activity. The antibacterial study shows that 3-Cyanopyridines derivatives showed moderate activity with MICs between 20 and 80 μg/mL. The 3-Cyanopyridines showed low antifungal activity. The importance of such work lies in the possibility that the new compounds might be more efficacious drugs against bacteria, which could be helpful in designing more potent antibacterial agent for therapeutic use.