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Synthesis, antimicrobial and antifungal activity evaluation of 2,6-diaryl-4-SECaminonicotinonitriles and 4-sec-amino-6-aryl2-(pyridin-2-YL)pyridine-3-carbonitriles i.e. Bipyridin... ARTICLE in THE JOURNAL OF PHARMACY · APRIL 2012

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Umesh D. Patil et al. / Journal of Pharmacy Research 2012,5(3),1383-1386

Research Article ISSN: 0974-6943

Available online through www.jpronline.info

Synthesis, antimicrobial and antifungal activity evaluation of 2,6-diaryl-4-SEC-aminonicotinonitriles and 4-sec-amino-6-aryl-2-(pyridin-2-YL)pyridine-3-carbonitriles i.e. Bipyridines and their docking study Umesh D. Patil, Ajay P. Nikum, Pramod S. Nagle and Pramod P. Mahulikar* *School of Chemical Sciences, North Maharashtra University, Jalgaon, 425001, India.

Received on:10-12-2011; Revised on: 15-01-2012; Accepted on:12-02-2012 ABSTRACT Herein, we present an innovative, new, greener and highly regioselective route for the synthesis of 2,6-diaryl-4-sec-amino-nicotinonitriles and 4-secamino-6-aryl-2-(pyridin-2-yl) pyridine-3-carbonitriles i.e. bipyridines, through base induced ring transformation. All the synthesised compounds were screen for their antimicrobial and antifungal activity and compounds were used to identify lead structure through the docking study approach by automated docking. This docking study is used to determine the orientation of inhibitors bound in the active site of glucosamine-6-phosphate synthase GlcN-6-P synthase (PDB code: 1JXA) to evaluate their antibacterial response. Key words: Nicotinonitriles, Pyridine-3-carbonitriles, Bipyridines, Antimicrobial Activity, Antifungal Activity, Molecular docking.

INTRODUCTION The worldwide resurgence of tuberculosis followed by the recent emergence of multi-drug resistant strains of Mycobacterium tuberculosis [1] has refocused attention towards this disease with a view to develop new and more effective antibacterial agents. The clinical management of acquired immune deficiency syndrome (AIDS) has become very challenging because the opportunistic infections, particularly Mycobacterium tuberculosis [ 2 ] and Mycobacterium avium[3] are more often responsible for the death of HIV-infected patients. Thus in order to control the rapid spread of tuberculosis, there is an urgent need to develop new antimycobacterial agents with unique modes of action to replace the current regimens. This preliminary communication demonstrates the in vitro antibacterial and antifungal activity of 2,6-diaryl-4-sec-amino-nicotinonitriles and 4-sec-amino-6-aryl-2(pyridin-2-yl)pyridine-3-carbonitriles i.e. bipyridines, which has not been disclosed so far. The objective of our study is to generate new leads and to optimize the structure to display the potent efficacy. The utility of pyridine rings in understanding the chemistry of biological system has been realized greatly because of their presence as substructures in many natural products[4] and pharmacologically active molecules [5] with wide synthetic potential for generating molecular diversity of therapeutic importance, and the ability to catalyze both biological and chemical reactions[4-7]. 4-Dimethylaminopyridine (DMAP), is a highly demanding reagent, used as a catalyst in acylation reactions and also for activation of carboxylic acids without racemisation of a chiral centres. Pyridines are excellent ligands for complexation with transition metals [7]. Alkene pendant pyridine polymers are industrially useful as acid scavengers[7] and as materials for chemical separations. The compounds containing pyridine ring possess a broad range of biological activities [7-9]. Pyridinyl-2-imidazoles have been shown to have dramatically decreased Cytochrome P450 binding

*Corresponding author. Pramod P. Mahulikar School of Chemical Sciences, North Maharashtra University, Jalgaon, 425001, India.

as compared to imidazoles with other substitution patterns[10], and also are potent, orally active anti-inflammatory agents[11]. On the other hand, the presence of the cyano group (-CN), on the pyridine ring, was essential for activity, like in a prion disease therapeutics [12], C-Jun NH2 terminal kinase (JNKs) inhibitors[13], orally active platelet-activating factor antagonists[14], etc. Similarly, the presence of cyano group (-CN) is widely used for transformation of it into amides, amines, esters, carboxylic acids, etc[15]. Hence they have been used as intermediates for the synthesis of fine chemicals such as agrochemicals, dyes and medicines [16]. Like substituted pyridines, 2,2'-bipyridines also establish a class of chelating hetero cyclic ligands[17]. Furthermore, the bipyridine core offers a myriad of possibilities in terms of modification by different substitution patterns which led to its manifold use as attractive building blocks in supra-, nanoand macromolecular chemistry [18-20] as well as, in the areas of analytical and photochemistry [21]. MATERIALS AND METHODS Chemistry The 2,6-diaryl-4-sec-amino-nicotinonitriles (5a-i) and 4-sec-amino-6-aryl2-(pyridin-2-yl)pyridine-3-carbonitriles (5j-l) were synthesized from a sequence of reactions[22] as depicted in Schemes 1 and 2. These synthesis are achieved through base induced ring transformation of suitably functionalized 4-sec-amino-2-oxo-6-aryl-2H-pyran-3-carbonitriles (2) with N-arylbenzamidines (3) or N-arylpicolinamidines (4) in DMF using KOH as the base, respectively. The various 4-sec-amono-2-oxo-6-aryl-2H-pyran-3-carbonitriles (2) used as precursors were prepared in two steps. The first step was the preparation of 4-(methylthio)-2-oxo-6-aryl-2H-pyran3-carbonitriles (1) from the reaction of ethyl 2-cyano-3,3bis(methylthio)acrylate and suitable acetophenones by using KOH in DMF[23]. And in second step amination of 1 with a cyclic secondary amine in refluxing methanol furnished 2. While, amidines (3 & 4) were prepared from the corresponding nitriles and amines [24]. All the synthesized compounds (Table-1) were characterized by their spectroscopic techniques and elemental analyses [22].

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Umesh D. Patil et al. / Journal of Pharmacy Research 2012,5(3),1383-1386 Scheme 1: Synthesis of 2,6-diaryl-4-sec-amino-nicotinonitriles (5a-i) from 2H-pyran-2one (2) and N-arylamidine (3).

SCH3 CN Ar

O 1

N CN

"Ar

3

NH 2

1

Ar

O 2

O

HN

KOH/DMF rt

Ar'

CN Ar

N Ar' 5a-i

3

Scheme 2: Synthesis of 4 -sec-amino-6-aryl-2-(pyridin-2-yl)pyridine-3-carbonitriles (5j-l) i.e. bipyridines.

Ar''

N CN

3

NH 2

1

HN Ar

O

N KOH/ DM F rt

N

O

CN Ar

N

Ar

R

Ar'

Ar''

Yields a (%)

5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l

5c

5d

5f m.p.

HN R C6 H 5 Piperidine C6 H 5 4-Br-C6 H 4 Piperidine C6 H 5 4-Br-C6 H 4 Morpholine C6 H 5 4-Cl-C6 H 4 Morpholine C6 H 5 4-Br- C6 H 4 Piperidine 4-Br-C6 H 4 4-Cl- C6 H 4 Piperidine C6 H 5 4-Cl- C6 H 4 Pyrolidine C6 H 5 4-CH 3 O-C6 H 4 Piperidine C6 H 5 4-Cl- C6 H 4 Pyrolidine 4-Br-C6 H 4 4-Br-C6 H 4 Pyrolidine 2-Pyridiyl 4-Cl-C6 H 4 Morpholine 2-Pyridiyl C6 H 5 Morpholine 2-Pyridiyl

5b

5e

Table 1: Synthesis of 2,6-diaryl-4-sec-amino-nicotinonitriles (5a-l)[22] . Entry

5a

N

5j -l

4

2

Entry No.

Concentrations µM

Zone of inhibition (dia. in mm) Staphylococcus Salmonella aureus typhimurium

Candida Albicans

75 125 250 500 75 125 250 500 75 125 250 500 75 125 250 500 75 125 250 500 75 125 250 500 75 125 250 500 75 125 250 500 75 125 250 500 75 125 250 500 75 125 250 500 75 125 250 500 75 125 250 500 75 125 250 500

2.1 8.3 9.6 13.6 16.2 3.4 2.2 4.2 8.7 9.3 11.4 15.3 6.1 8.3 9.9 13.2 4.3 21.4 26.6 29.3 34.8 -

7.6 9.53 15.8 21.6

N

N H

O

Table 2: Antibacterial and antifungal activity study of synthesised compounds 5a-l.

5g 4-F-C6 H 4 C6 H 5 4-F-C6 H 4 4-F-C6 H 4 H H 5-Br H H 4-Br-C6 H 4 4-Br-C6 H 4 3,4-Cl-C6 H 3

48% 48% 46% 39% 22% 16% 22% 38% 44% 43 % 48 % 52 %

o

160 C 173 o C 172 o C 198 o C 168 0 C 172 0 C 156 0 C 166 0 C 122 0 C 208 o C 166 o C 154 o C

a – isolated yields by column chromatography.

5h

5i

5j

5k

BIOLOGICAL ACTIVITY STUDY Antimicrobial and Antifungal Activity Evaluation of the Synthesised Compounds Herein the synthesized compounds (5, a-l) were screened for their antibacterial and antifungal activity[25] against both Staphylococcus aureus (gram positive) and Salmonella typhimurium (gram negative) bacteria and Candida Albicans fungous species. The concentration of the test compounds used was 500, 250, 125 and 75 µM. Cephalexin and Nyastatin were used as standard compound. Preparation of Culture Medium and Inoculation: For antibacterial activity, 35 g of nutrient agar and 10 g of agar-agar were suspended in distilled water (1000 mL) by boiling. Media and Petri dishes were sterilized in autoclave at pressure 15 lbs for 20 minutes. Under aseptic condition, 20 mL of media was dispended into sterilized Petri dishes to yield a uniform depth of 6 mm. After solidification of the medium, the bacterial culture was inoculated by spread plating technique. Disc Application and Incubation: Discs of 6 mm diameter were prepared from Whatmann No. 1 filter paper, sterilized by autoclaving and subsequently dried at 800 C for an hour. The sterilized discs were immersed in respective formulations of compounds, and placed on nutrient agar surface with flamed forceps and gently pressed down to ensure contact with the agar surface. The disc was spaced for enough to avoid both reflection waves from the edges of Petri dishes and overlapping rings of inhibition. Finally, the Petri dishes were incubated for 24 h at 37 0 C in an inverted position. After 24 h the diameter (mm) of the inhibition zone around each spot was measured. Antibacterial activities were indicated by clear zone of growth inhibition. All experiments were performed twice and averages of the results were considered (Table 2).

5l

Cephalexin

Nyastatin

3.2 3.8 3.9 4.4 4.1 6.8 8.8 10.9 14.7 8.4 9.9 13.7 16.8 5.4 7.2 9.7 12.5 22.7 27.3 31.1 35.5 -

Note:’-‘ indicates no zone of inhibition.

Molecular docking studies: The docking study was performed by automated docking and were used to determine the orientation of inhibitors bound in the active site of glucosamine-6-phosphate synthase GlcN-6-P synthase (PDB code: 1JXA) for antibacterial. The ligands were drawn in ChemDraw Ultra 6.0. The Hex 6.1 automated docking programme was used to perform molecular docking. The docking of ligand molecules with GlcN-6-P synthase revealed that all the inhibitor compounds exhibited the bonding with one or more amino acids in the active pocket as shown in (Fig. 1) and it is cased between the GLU 79, GLY78, HIS77, GLU534, SER 531, GLU 121, ARG 108, HIS 104, GLU 105 amino acids and therefore it may be considered as good inhibitor of GlcN-6-P synthase. The study also revealed that the 2,2’bipyridine scaffold is the key residues at the active site of GlcN-6-P synthase. Moreover many compounds have minimum binding and docking energy hence could be considered as a good inhibitor of GlcN-6- P. In all the synthesized compounds, 5h compound show highest energy score whereas 5l shows lowest energy score. The theoretical outcome highlighted that the

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Umesh D. Patil et al. / Journal of Pharmacy Research 2012,5(3),1383-1386 minimum binding energy of the molecules with the targeted protein may make these newly synthesized 2,2’-bipyridine compounds as good inhibitors to glucosamine-6-phosphate synthase. Therefore it is pleasing to state that the docking studies have widened the scope of developing 2,2’-bipyridine as promising antibacterial agents.

A)

Electrostatic Density Map: The electrostatic potential is potential energy felt by a positive “test” charge at a particular point in space. If the ESP is negative, this is a region of stability for the positive test charge. Thus, an ESP- mapped density surface can be used to show region of a molecule that might be more favourable toward the +ve or –ve area of enzyme. Determination of the electronic properties of compounds was performed using the program ArgusLab 4.0.1. This program selects points on the 0.002 isodensity surface au (atomic units) and shows the surface of the map on a scale of colours, each colour represents the different electrostatic potential of the molecule. The most positive electrostatic potential is shown in white, followed by a scale of tones, the red colour indicating the most negative electrostatic potential. The electrostatic density maps of two representative maps are presented in Fig. 2 for compound 5a and 5f. It is evident from these figures that the regions containing 2,2’-Bipyridine rings exhibited comparatively high electron density regions and high susceptibility toward the receptor.

B)

Figure 2: Electrostatic potential of the compound 5a and 5f

C)

D)

E)

F)

Figure 1: Binding mode of inhibitors in active site of GlcN-6-P. A) Interaction of 5c with GlcN6-P. B). Binding of 5c with amino acids of GlcN-6-P. C) Interaction of 5h with GlcN-6-P. D) Binding of 5h with amino acids of GlcN-6-P. E) Interaction of 5i with GlcN-6-P. F) Binding of 5 i with amino acids of GlcN-6-P. Sample Code

E total (KJ/ Mol)

5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l

-146.18 -154.00 -150.69 -148.51 -129.61 -158.81 -125.08 -161.24 -113.49 -121.38 -110.54 -108.47

RESULTS AND DISCUSSION In summary, a green, novel, short, highly regioselective and efficient synthetic protocol for 2,6-diaryl-4-sec-amino-nicotinonitriles (5a-i) and 4-secamino-6-aryl-2-(pyridin-2-yl)pyridine-3-carbonitriles (5j-l) have been developed. As an illustrative application of this strategy, a symmetrical, unsymmetrical and heteroaryl derivatives of 2,6-diaryl-4- sec -aminonicotinonitriles and 4-sec -amino-6-aryl-2-(pyridin-2-yl)pyridine-3carbonitriles i.e. highly substituted unsymmetrical 2,2'-bipyridines were prepared easily. Our objective was to design pyridine and bipyridine based compounds to identify lead structures through the docking study approach by automated docking and used to determine the orientation of inhibitors bound in the active site of glucosamine-6-phosphate synthase GlcN-6-P synthase (PDB code: 1JXA) to evaluate their antibacterial response. In the antifungal activity evaluation study, all synthesised compounds 5a-l, were found to be almost inactive, against Candida Albicans as a fungus species. In antibacterial activity evaluation study, all synthesised compounds 5a-l, were found to be almost inactive, against both test bacterial species. Only few compounds (5a, d, j, k and l) exhibited poor antibacterial activity in comparison with standard. REFERENCES [1] Davidson PT, Le HQ, Drug treatment of tuberculosis – 1992, Drugs, 1992, 43, 651-673. [2] For an excellent review of the tuberculosis area, see: Bloom, B. R., Ed. Tuberculosis: Pathogenesis, Protection, and control; ASM: Washington, DC, 1994. [3] Horsburgh CR, Jr MD, Mycobacterium avium Complex Infection in the Acquired Immunodeficiency Syndrome, Engl. J. Med. 1991, 324, 1332-1338. [4] Joule JA, Smith G, Mills K, Heterocyclic Chemistry, 3 rd ed, Chapman and Hall: London, 1995, 72–119. [5] Roth HJ, Kleeman A, Pharmaceutical Chemistry, Drug Synthesis, Prentice Hall Europe: London, 1988, Vol.1, 407.

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Umesh D. Patil et al. / Journal of Pharmacy Research 2012,5(3),1383-1386 [6] MDDR: MDL, Drug Data Registry, by MDL informations Systems: San Leandro, California, USA. [7] Vacher B, Bonnaud B, Funes F, Jubaudt N, Koek W, Assie MB, Cosi C, Kleven M, Novel Derivatives of 2-Pyridinemethylamine as Selective, Potent, and Orally Active Agonists at 5-HT1A Receptors J. Med. Chem.1999, 42, 1648-1660. [8] Agrawal KC, Sartorelli AC, Relationship between Structure and Antineoplastic Activity of Arylsulfonylhydrazones of 2-Formylpyridine N-Oxide J. Med. Chem., 1978, 21, 218-221. [9] Kawamura S, Hamada T, Sato R, Sanemitsu Y, 2,6-Diphenylpyridines: A New Class of Herbicides, J. Agric. Food Chem. 1991, 39, 22792281. [10] Murray M, Wilkinson CF, Interactions of nitrogen heterocycles with cytochrome P-450 and monooxygenase activity, Chem. Biol. Interact., 1984, 50, 267-275. [11] Khanna IK, Yu Y, Huff RM, Weier RM, Xu X, Koszyk FJ, Collins PW, Cogburn JN, Isakson PC, Koboldt CM, Masferrer JL, Perkins WE, Seibert S, Veenhuizen AW, Yuan J, Yang D-C, Zhang YY, Selective Cyclooxygenase-2 Inhibitors: Heteroaryl Modified 1,2Diarylimidazoles Are Potent, Orally Active Antiinflammatory Agents, J. Med. Chem. 2000, 43, 3168-3185. [12] Reddy TRK, Mutter R, Heal W, Guo K, Gillet VJ, Pratt S, Chen B, Library Design, Synthesis, and Screening: Pyridine Dicarbonitriles as Potential Prion Disease Therapeutics J. Med. Chem. 2006, 49, 607-615. [13] Zhao H, Serby MD, Xin Z, Szczepankiewicz BG, Liu M, Kosogof C, Liu B, Nelson LTJ, Johnson EF, Wang S, Pederson T, Gum RJ, Clampit JE, Haasch DL, Zapatero C-A, Fry EH, Rondinone C, Trevillyan JM, Sham HL, Liu G, Discovery of Potent, Highly Selective, and Orally Bioavailable Pyridine Carboxamide c-Jun NH2Terminal Kinase Inhibitors, J. Med. Chem. 2006, 49, 4455-4458. [14] Carceller E, Merlos M, Giral M, Balsa D, Rafanell JG, Forn J, Design, Synthesis, and Structure-Activity Relationship Studies of Novel 1-[(1-Acyl-4-piperidyl) methyl]-1H-2-methylimidazo[4,5-

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Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.5 Issue 3.March 2012

1383-1386