Synthesis, Molecular Docking and Antibacterial Studies of Novel Azole derivatives as Enoyl ACP Reductase Inhibitor in Escherichia coli.

 

Sindhu. T. J1*, Arathi. K. N, Akhila Devi, Aswathi. T. A, Noushida. M, Midhun. M, Sajil Saju Kuttiyil

Department of Pharmaceutical Chemistry, Sanjo College of Pharmaceutical Studies, Vellappara, Palakkad.

*Corresponding Author E-mail: sindhutj81@gmail.com

 

ABSTRACT:

The current research is aimed to design of enzyme inhibitors as target for the drug discovery. Azoles  can inhibit the enoyl ACP reductase that present in the E. coli is an essential bacterial enzyme. Fabl, the enoyl acyl carrier protein reductase of the Escherichia coli species is one of the attractive targets in E. coli associated diseases. By considering the above observations, an attempt is made here to design and synthesize various N-Mannich bases of azoles as enoyl ACP reductase inhibitors.The synthesized mannich bases were subjected to molecular docking studies with enoyl ACP reductase  (PDB ID: 1C14) by using in silico studies by molinspiration online tool. These new compounds were evaluated for their antimicrobial activity. The compounds showed good activity against bacteria Escherichia coli comparable to that of standard drugs Ciprofloxacin. All newly synthesized compounds A1-5 showed antifungal activity towards the tested clinical strain of Escherichia coli. Among these, N-Mannich base compound A3 (benzimidazole derivative) showed good zone of inhibition. In Molecular docking studies, Among the synthesized compounds, diphenylamine derivative of Benzotriazole (A1) has shown  binding energy (–5.87 kcal/mol) target protein.

 

KEYWORDS: Enoyl ACP Reductase Inhibitors, Antimicrobial Activity, Mannich Base, Molecular Docking, Binding Energy.

 

 


INTRODUCTION:

The drugs that we use in our day to day life, in order to explore the physiological systems and to treat any pathological conditions are mainly chemically synthesized compounds. Although there are a number of sources from which we can obtain medicaments like natural sources, (plants and animals), we mainly depend on synthetically produced new drugs those which are synthetically altered for superior performances in the biological system with minimum or without side effects(1)

 

 

DRUG DESIGN AND DISCOVERY:

Drug discovery and development is an intense, interdisciplinary and endeavour, when a compound is under taken for discovery. It involves synthesis, characterisation, screening and evaluation for therapeutic efficiency. Azoles are a class of five-membered heterocyclic compounds containing a nitrogen atom and at least one other non-carbon atom (i.e. nitrogen, sulfur, or oxygen) as part of the ring [2,3]. Their names originate from the Hantzsch–Widman nomenclature[4]. The azoles that are available for systemic use can be classified into two groups: Triazoles (eg: Fluconazole, Itraconazole, Voriconazole, Posaconazole, and Isavuconazole) and the Imidazoles (eg: ketoconazole)[5]. Other systemic antifungal agents, such as amphotericin B and flucytosine, are discussed separately.  Azole antifungal agents can be used to treat fungal infections of the body and skin, including athlete's foot, onychomycosis, ringworm, and vaginal candidacies. Antifungals can be grouped into three classes based on their site of action: azoles, which inhibit the synthesis of ergosterol (the main fungal sterol); polyenes, which interact with fungal membrane sterols physicochemically; and 5-fluorocytosine, which inhibits macromolecular synthesis.(6)

 

MANNICH REACTION:

Mannich reaction is an organic reaction which consists of an amino alkylation of an acidic proton placed next to a carbonyl functional group by formaldehyde and a primary or secondary amine or ammonia[7]. The final product is a β-amino-carbonyl compound also known as a Mannich base[8] Figure No:1). Reactions between aldimines and α-methylene carbonyls are also considered mannich reactions because these imines form between amines and aldehydes. The reaction is named after chemist Carl Mannich [9,10,11].

 

Figure No: 1   General mannich base reaction

 

The project aims at synthesizing mannich bases of different azoles using diphenylamine as secondary amine, their characterization. The mannich base of phenytoin using the same secondary amine is also synthesized and characterized. The biological activities of azoles like Benzotriazole, 1, 2, 3, 4-Tetrahydro carbazole, Benzimidazole and 3,5- Dimethyl Pyrazole are determined and then compared with that of Phenytoin. The synthesized compounds were theoretically tested for their biological activity using molecular docking studies. The experimentally determined biological activity was again compared with that obtained theoretically.

 

MOLECULAR DOCKING STUDIES:

In the field of molecular modeling, docking is a method which predicts the orientation of one molecule to a second when bound to each other to form a stable complex. Preferred orientation helps to predict the strength of association or binding affinity between two molecules. The associations with biological molecules such as proteins, nucleic acids, carbohydrates and lipids play an important role in signal transduction. The orientation of the interacting molecules decides the signal transduction i.e. agonism or antagonism. So docking is an useful tool for predicting both the strength and type of signal produced. A new series of mannich bases of Benzimidazole, Benzotriazole, 3,5-dimethyl pyrazole, 1,2,3,4-tetrahydro carbazole and phenytoin have been synthesized by mannich base reaction between the mentioned compounds, formaldehyde and diphenylamine. Structure of these compounds was established by IR, 1H NMR and mass spectroscopy [12]. The synthesized mannich bases were subjected to molecular docking studies. These new compounds were evaluated for their antimicrobial activity[13]

 

EVALUATION OF BIOLOGICAL ACTIVITY:

Primarily, the anti microbial activities of the synthesized compounds were tested against E.coli using Disk diffusion method at a concentration of 200μg. The test compounds were compared with standard disc of Ciprofloxacin. Solvent dimethyl formamide (DMF) was kept as a control.

 

MATERIALS AND METHODS:

PHASE 1:IN-SILICO METHODS

Softwares and databases used

v AutoDock 4.2 combines

§  AutodockTools1.5.4

§  Python Molecule viewer1.5.4

§  Vision 1.5.4

v Python 2.5

v Accelrys discovery studio viewer

v Pre ADMET software

v Molinspiration server

v RCSB protein data bank

v Online SMILES translator         

 

All the in-silico experiments are carried out at Sanjo College of Pharmaceutical Studies, vellapara, palakkad.

 

The leads selected were substituted with various substituents and they were optimized for the pharmacokinetic parameters by evaluating. The in-vivo absorption capabilities of the designed molecules were assessed by the means of Lipinski’s rule of five using molinspiration server. All the lead compounds satisfied the rule indicating that the ligands A1-5 have good oral absorption. (Table No:1)


 

 

Table No: 1 Drug likeness scores of A1-5 using molinspiration server

Sl. No

Compound code

mLogP

MW

No. of H acceptors

No. of H donors

No. of violations

01

A1

4.77

300.37

4

0

0

02

A2

6.82

352.48

2

0

1

03

A3

4.91

299.38

3

0

0

04

A4

5.83

433.51

5

1

1

05

A5

4.93

276.38

2

0

0


DOCKING STUDIES FOR THE LEAD MOLECULES:

After the lead has been optimized, the protein was subjected to docking studies using AutoDock 4.2 for evaluating the binding interactions. (14,15,16,17)

 

Enzyme and ligand preparation:

The X-ray crystal structure of the enzyme enoyl ACP reductase (FabI) of E. coli (PDB entry: 1C14) was obtained from Protein Data Bank (http://www.rcsb.pdb.org). (18)

 

SELECTION OF Enoyl-[acyl-carrier-protein] reductase FROM PDB : E coli enoyl reductase-NAD+-triclosan complex (1C14)

Escherichia coli

PDB accession code is 1C14

Resolution  is 

Chains A, B

Sequence Length  262

 

PHASE II: SYNTHESIS AND CHARACTERIZATION:

Chemicals and reagents used:

O-phenylenediamine, formic acid, sodium hydroxide, charcoal, Dimethyl formamide, diphenyl amine, acetyl acetone, ferric chloride, hydrazine hydrate, benzimidazole, 1,2,3,4-Tetrahydrocarbazole, Phenytoin and benzotriazole. The chemicals used were obtained from Kerala Scientific, Palakkad, Chemind Chemicals, Palakkad and Medwin Diagnosis, Malappuram. All the compounds were purified and dried whenever necessary before use.

 

Apparatus used:

Beakers, test tubes, glass rods, magnetic stirrer, thermometer, round bottom flask, reflux condenser, funnel, conical flasks, separating funnel, dropping funnel and pipettes.

 

Analytical work:

Melting points were determined by using melting point apparatus and were uncorrected. Reactions were monitored by thin layer chromatography (TLC) on pre-coated silica gel G plates using Iodine vapour as visualizing agent.

 

UV spectra were recorded on ELICO Double Beam SL 210 UV VIS Spectrophotometer in the Department of Pharmaceutical Analysis, Sanjo College of Pharmaceutical studies.

 

PREPARATION OFAZOLES:

Preparation of 1,2,3,4 –tetrahydocarbazole(19,20)

Take 2ml of cyclohexanone and 12ml of glacial acetic acid in round bottom flask. Reflux with frequent shaking and add slowly 2gm of phenyl hydrazine with caution. Heat the mixture for 20 minutes under reflux. Cool with vigorous shaking. Filter the brown product, wash with cold water and recrystallize from alcohol [28] Figure No:2.

 

Figure No:2      Preparation of 1,2,3,4,-Tetrahydrocarbazole

 

Preparation of 3,5-dimethyl pyrazole(21)

Take 5 ml acetyl acetone with 1 ml freshly prepared ferric chloride solution in a beaker until an orange red precipitate is formed. A cold solution of hydrazine hydrate in 20ml dilute sodium hydroxide with continuous stirring. The temperature is kept below 15°C. The solution is stirred for 1 hour at 20°C. Cool the solution and collect the precipitate. It is filtered, washed with cold water and collected [27]. (Figure No:3)

 

Figure No: 3 Preparation of 3,5-dimethyl pyrazole

 

PREPARATION OF BENZOTRIAZOLE(22,23)

Dissolve 5.4gm of o-Phenylenediamine to a pure mixture of 6ml acetic acid in 15ml water containing beaker. Warm slightly if necessary. Cool the clean solution to 15°C.  Stir vigorously then add a solution of 3.25gm of sodium nitrite in 5ml of water in small portion. The reaction mixture becomes warm within 2 to 3 minutes.  The temperature reaches 85°C and begins to cool. While the colour changes from deep red to pale brown stir for 15 min. Cool up to 35-40°C thoroughly chill in ice water bath for 30 min. The pale brown solid separated by filtration and washed with ice cold water. Dissolve in 65 ml boiling water and add charcoal to boil for 5 min. Filter and allow the filtrate to cool below 60°C by adding few crystals of crude benzotriazole which have been retained by seeding. Allow the mixture to attain room temperature slowly and thoroughly chilled in ice and collect benzotriazole, which seperates as pale straw colour needles[27]. (Figure No:4)

 

Figure No:4    Preparation of Benzotriazole

 

 

PREPARATION OF BENZIMIDAZOLE(24,25)

Place 10 g of o-phenylenediamine in a round bottom flask of 250 ml add 6.5ml 90%Formic acid. Heat the mixture in a water bath at 100°c for 2 Hour. cool and add10% NaOH solution slowly, with a constant rotation of the flask, until the mixture is just alkaline to litmus. Filter off the synthesized crude benzimidazole by the pump, wash with ice cold water, drain well and wash again with 25ml of cold water. Dissolve the synthesized product in 400ml of boiling Water. Add 2g charcoal and digest for 15 min. Filter rapidly. Cool the filtrate to 10°c, filter off the Benzimidazole. Wash with 25ml coldwater, dry it in 100°c[11].

 

Figure No:5    Preparation of Benzimidazole

 

PREPARATION OF PHENYTOIN28

Place 5.3g of benzil, 3g of urea. 15ml 30%   NaoH solution and 75ml ethanol in a 100ml round bottom flask. Attach a reflux condenser and boil under reflux for at least 2 hours. Cool at room temperature; pour the reaction product in to 125ml of water. Mix thoroughly allows standing for 15 minutes and filter to remove any insoluble byproduct. Render the filtrate strongly acidic with concentrated hydrochloric acid. Cool in ice water and immediately filter out the precipitated product. Recrystallize the product from spirit .

 

Figure No:6   Preparation of phenytoin

 

PREPARATION OF MANNICH BASE FROM   AZOLES:

FROM 1, 2, 3, 4-TETRAHYDROCARBAZOLE (A2)

To a solution of 1,2,3,4-Tetrahydrocarbazole of 0.01mol in DMF, formaldehyde, 0.6 ml, was added under stirring. The reaction mixture was stirred at room temperature for 30 minutes. To complete the reaction of formaldehyde and to yield methylol derivative, to this solution, 2.75 ml 2° amine was added drop wise and refluxed for 2hours. The reaction mixture was poured into ice-cold water and filtered and washed with hot water finally it was dried and purified by recrystallization from acetic acid.

 

Figure No:7  Mannich base of 1,2,3,4-tetrahydro carbazole

 

FROM 3,5- DIMETHYL PYRAZOLE (A5):

Dissolve 0.96 gm 3,5-dimethyl pyrazole in DMF and add 0.5 ml formaldehyde in a round bottom flask. It is refluxed for 2 hours. The solution is then treated with 3.31 ml of diphenyl amine and stirred for 30 minutes. Transferred the solution to ice cold water and stirred continuously. The precipitate was filtered washed and recrystallized from ethanol.

 

Figure No:8 Mannich base of 3,5-dimethyl pyrazole

 

FROM BENZOTRIAZOLE (A1):

To a solution of 1.19g of benzotriazole in DMF add 0.6ml of formaldehyde. The Reaction mixture was stirred at room temperature for 30 minutes. The solution is then added with 2.75ml of diphenylamine and  refluxed for 2 hours. The reaction mixture was poured in to ice cold water and filtered and washed with cold water. Finally it was dried and purified by recrystallization from acetic acid.

 

Figure No:9 Mannich base of benzotriazole

 

FROM BENZIMIDAZOLE (A3):

To a solution of benzimidazole 0.01 mol in DMF formaldehyde (0.6 ml) was added under stirring. The reaction mixture was stirred at room temperature for 0.5 hour to complete reaction of formaldehyde end to yield methylol derivative. To this solution, 0.02 mol of secondary amine was added drop wise and refluxed for 2 hour. The reaction mixture was poured into ice cold water  and filtered  and  washed  with  hot  water. Finally it was dried and   purified by recrystallization from acetic acid.

 

 

PREPARATION OF MANICH BASE FROM PHENYTOIN (A4)

To a solution of 2.52g of phenytoin in DMF add 0.6ml of formaldehyde The Reaction mixture was stirred at room temperature for 30 minutes. The solution is then added with 2.75ml of diphenylamine and refluxed for 2 hours. The reaction mixture was poured in to ice cold water and filtered and washed with cold water. Finally it was dried and purified by recrystallization from acetic acid.

 

 

Figure No:11 Mannich bases of Phenytoin

 

PHASE 3:ANTI-BACTERIAL STUDIES(33-39)

 

APPARATUS AND CHEMICALS REQUIRED

Sterile swab          :               Hi Media

Non-absorbent cotton: Rama Raju Surgical cotton Ltd.

Conical flask:       Borosil  

Test tubes:            Borosil

Petri dishes:          SD Fine – Chem Ltd.

Micropipettes:      VARI pipettes (Hi – Tab Lab)

Autoclave:            Universal Autoclave

Laminar air Flow unit:       CLEAN AIR Instruments Inc.

Micro tips:            Tarsons

 

The antibacterial screening was carried out in the Pharmaceutical Microbiology laboratory, Sanjo college of Pharmaceutical Studies, vellapara, palakkad.

 

 

 

 

PROCEDURE FOR ANTIBACTERIAL ACTIVITY

The sterilized (autoclave at 1200c for 30 minutes) nutrient agar medium (40-500c) was inoculated with the suspension of microorganism and mixture was transferred to sterile petridishes and allowed to solidify. In each plate 10mm disc was placed. Ciprofloxacin disc of conc. 1µg placed kept as standard and 20µl synthesized compounds (200µg/µl) solutions in DMF is poured. The plates were kept in refrigerator for 30 minutes to allow the diffusion of sample to the surrounding agar medium. The plates were incubated at 37±20c for 24 hours and observed for antibacterial activity. The diameter of zone of inhibition were measured and compared with that of standard. The values were tabulated.

 

Method: Agar Disk Diffusion Method

Test organism: E. coli

Standard drug: Ciprofloxacin (1µg/ml)

Control: Dimethyl formamide

Concentration: 200 µg/ml

Culture medium: Nutrient agar medium

 

RESULTS AND DISCUSSION:

DOCKING STUDIES

The results of docking of Enoyl-[acyl-carrier-protein] reductase (1C14.pdb) with the ligands A1-5 are reported below. The best docked structures should have the binding energy higher than that of the standard. The binding sites were represented in the snap shots and the binding energy was compared with the standard ligand, ciprofloxacin. The results are mentioned in the table  followed by the snapshots.

 

Table No:2 Binding energies of A1-5 with enoyl ACPreductase (1C14.pdb)

Sl. No

Compound Code

Binding energy (∆G = kcal/mol)

01

A1

-5.87

02

A2

-5.63

03

A3

-5.13

04

A4

-5.42

05

A5

-4.81

06

Ciprofloxacin

-5.45

 

 

Figure No:12

Snapshots and binding interactions of standard Ciprofloxacin with enoyl acp reductase (Binding Energy= -5.45)

 

Figure No:13

Snapshots and binding interactions of mannich bases of benzotriazole with enoyl acp reductase (A1 binding energy= -5.87)

 

In N-Mannich base of azole series all the compound shown excellent binding interaction. Most of the derivatives were interacting with the key active site of the enoyl ACP reductase with superior binding energy. Among the synthesized compounds, diphenylamine derivative of Benzotriazole (A1) has shown least binding energy–5.87 kcal/mol compared to standard ciprofloxacin (-5.45 kcal/mol).

 

Other derivatives such as A2 and A4 also showed very good binding energy -5.63 kcal/mol and -5.42 kcal/mol  respectively. But compound A3 and A5 showed highest binding energy-5.13 kcal/mol and -4.81 kcal/mol respectively.

 

CHARACTERIZATION OF SYNTHESIZED MANNICH BASES OF AZOLES

The novel 5 docked compounds (A1-5) were synthesized by the schemes as depicted above, and the structures of the synthesized compounds were established on the basis of their physical data (Melting Point and TLC) and spectral data UV[29,30,31,32].

 

Recrystallization solvent:  Alcohol

Solvent system     :  Acetone: Benzene (3:7)

 Visualizing agent:  Iodine vapour

 

Table No:3 Physical characteristcs of synthesized compounds

Compound code

Chemical Name

Mol.Formula

Mol. wt

Melting Point

Rf Value

Per. Yield%

A1

N, N-di phenyl-1H-benzotriazol -1-amine

C19H16N4

300.37

61° -62°

0.90

67.16

A2

N-(1,2,3,4-tetrahydrocarbazol-1ylmethyl)-N-diphenyl amine

C25H24N2

 

352.48

oily

0.83

62.87

A3

N-(1H-benz imidazol-2-yl methyl)-N-phenyl aniline

C20H17N3

299.38

64°-65°

0.83

45.54

A4

N-(5,5-di phenyl hydantoin-1-yl methyl) -N-diphenyl amine

C28H23N3O2

 

433.51

oily

0.84

77.29

A5

N-[(3,5-di methyl-1H-pyrazol -1-yl) methyl] -N-phenyl aniline

C18H19N3

276.38

71°-72°

0.92

68.48

 


Table No:4 Maximum absorbance of UV by A1-5 And its solubility

Comp.

Solubility

λ Max

(nm)

Water

Alcohol

DMSO

CHCl3

A1

In

Sol

Sol

Sol

320

A2

In

Sol

Sol

Sol

320

A3

In

Sol

Sol

Sol

320

A4

In

In

Sol

Sol

330

A5

Sol

Sol

Sol

In

320

Sol-soluble; In- insoluble

 

ANTIBACTERIAL ACTIVITY:

The zone of inhibition of 5 synthesised compounds are shown in Figure and their diameters were compared with that of the standard ciprofloxacin mentioned in Table No:5

 

Table No:5 The zone of inhibition of synthesised compounds Escherichia coli

Compound Code

Zone of Inhibition (mm)

A1

1

A2

1

A3

4

A4

3

A5

2

Ciprofloxacin

10

Control -DMF

1.5

 

 

Figure No :14 Antibacterial Screening of test compounds Escherichia coli

 

 

CONCLUSION:

Computer aided drug design helps to minimise the tedious drug discovery            process over the traditional method.

 

In silico ADME study and drug likeness score of the ligands observed helped to predict a better pharmacokinetic activity and oral bioavailability of the designed leads.

 

The binding energies obtained from docking study of  enoyl ACP reductase confirms that the lead compound inhibit the enzymes present in Escherichia coli.

 

Designed schemes were utilised in the preparation of novel azoles incorporated N-mannich bases of secondary amines.

 

The screening of synthesized compounds for antibacterial study      revealed that N-mannich bases of azoles and phenytoin show antibacterial activity.

 

Thus the present study depicts that the utilization of computer aided drug design is an efficient tool in predicting the effectiveness of a series of compounds under study and thus can result in the design of potent antibacterial agent. Although CADD is an efficient tool in determining the efficiency of chemical moieties, the data obtained from the docking studies in the present study comply with the result obtained from antimicrobial studies conducted at least at this concentration.

 

Further studies using increased concentrations may prove otherwise. This study   confirms that the lead compound inhibit the enzymes present in Escherichia coli.

 

REFERENCES:

1.      K.D. Tripathi, Essentials of medical Pharmacology, seventh edition, page no: 77-81

2.      J. A Joule and K. Mills, Heterocyclic Chemistry, 5th edition page no: 557-558.

3.      Alan R Katritzky and M. Lagowski, The principles of heterocyclic chemistry page no:145-146.

4.      I.L Finar, Organic Chemistry, Steriochemistry and chemistry of natural products, Volume 2, Fifth edition ,pg.no:622-647

5.      Prof. Ramarao Nadenda, medicinal chemistry, 2nd edition, pharma med press publications, page no:50-52,204.

6.      K. Ilango and P. Valentina, Textbook of medicinal chemistry, vol 1, Pg no. 177-178

7.      F.M Mann and B.C Saunders, Practical organic chemistry, Fourth edition, pg.no:261-262

8.      Gurdeep Raj, organic name reactions and Molucular rearrangements, page no:226 -228.

9.      V.K Ahluwalia and Rakesh kumar, Organic reaction mechanisms, Fourth edition, pg.no:304,352

10.   Thomas L. Lemke, David. A Williams, Victoria F. Roche, S. Wiliam Zito, Foye’s Principles of medicinal chemistry, seventh edition, pg.no:29-30

11.   L. Racane, V. T. Kulenovic, L. F. Jakic, D. W. Boykin, and G. K. Zamola, “Synthesis of bis-substituted amidino-benzothiazoles as potential anti-HIV agents,” Heterocycles, , 2001.55, 2085– 2098

12.   Ernest L. Eliel, Samuel H. Wilen and with contribution by Lewis N Mander, Steriochemistry of organic compounds pg.no :40

13.   John. M. Beale Jr., John H. Block, Wilson and Gisvold’s, Textbook of organic and medicinal and Pharmaceutical chemistry, 12th edition, Pg no. 25,215.

14.   Helmut B, Petra W, Angela ET, Birgit L et al. Protein EnvM Is the NADH-dependent enoyl-ACP Reductase (FabI) of Escherichia coli. J Biol Chem. 1994; 269: 5493-96.

15.   Richard JH, Charles OR. Enoyl-acyl carrier protein reductase (fabI) plays a determinant role in completing cycles of fatty acid elongation in Escherichia coli. J Biol Chem. 1995; 270: 26538-42.

16.   Roujeinikova A, Levy CW, Rowsell S, Sedelnikova S, Crystallographic analysis of Triclosan bound to enoyl reductase. J Mol Biol. 1999; 294: 527-35.

17.   Design, docking, synthesis and anti-E. coli screening of novel thiadiazolo thiourea derivatives as possible inhibitors of Enoyl ACP reductase (FabI) enzyme, Sonia George, Ramzeena Mohammed Basheer, Sayee Vignesh Ram, Senthil Kumar Selvaraj, Shinu Rajan and Thengungal Kochupappy Ravi DOI :10.3329 /bjp.v9i1. 16992, J Pharmacol. 2014; 9: 49-53.

18.   http://www.rcsb.pdb.org

19.   Hala Bakr, EL Nassan- Synthesis and antitumor activity of tetrahydrocarbazole hybridized with dithioate derivatives, Published- 05 Jun 2014 volume.

20.   Biswanath chakraborty, study the antibacterial activity of 1,2,3,4 tetrahydrocarbazole derivatives, volume may 2014, 8 pages, department of biochemistry of medical biotechnology

21.   Swaroopa H.M- synthesis and characterisation of some new pyrazole analogues for antimicrobial activity, published- 2011, page no 4-5

22.   Yu Ren, Ling Zhang, Cheng-HeYu Ren, Ling Zhang, Cheng-He -Development of Benzotriazole-based Medicinal Drug, Published - August 20, 2014

23.   Khalid A. Agha, Aader E. Abo-Dya, Tarek S. Ibrahim, eatadal H. Abdel-aal and Wael A. Hegazy-benzotriazole mediated synthesis and antibacterial activity of novel N-acylcephalexin-2016

24.   Yogita Bansal, Om Silakari-The therapeutic journey of benzimidazoles, Published in 2012.

25.   Shatha Ibrahim Alaqeel-Synthetic approaches to benzimidazoles fromo-phenylenediamine, published-13 August 2016

26.   Uthumporn Kankeaw and Ratchaneeporn Rawanna The Study of Antibacterial Activity of Benzimidazole Derivative Synthesized from Citronellal, Department of Chemistry, Faculty of Science, April 13, 2015;

27.   Vogel’s textbook of practical organic chemistry, 5th edition, Pg.no: 1153

28.   Organic chemistry, volume-1, 6th edition by I.L. Finar, Pg.no: 846, 670

29.   P C. Kamboj, Pharmaceutical Analysis 3, Instrumental methods, Spectroanalytical and Electoanalytical, Ultraviolet/ visible molecular absorption spectroscopy, Pg no. 103,123.      

30.   Egon Skehl, Thin Layer Chromatography a laboratory Handbook, 2nd edition fully revised and expanded, Pg no. 52, 66.

31.   Dr. S. Revisankar, Textbook of Pharmaceutical Analysis, 4th edition Pg no. 14.2- 14.6.

32.   Jerry March, Advanced organic chemistry, Reactions, Mechanisms and Structures, fourth edition, pg. no: 900- 902

33.   Antimicrobial activity of some new azole compounds by S Khabnadideh, Z Rezaei, Y Ghasemi, N Montazeri, Najafabady, Anti infective agents, Issue-1, Volume 10, 2012.

34.   Michael J. Pelczar, JR, E.C.S CHAN and Noel R. Krieg, Microbiology- Disc Plate method, 6th edition, Pg no. 535-537.

35.   N.K Jain, Pharmaceutical Microbiolog, Rivised and updated second edition, Vallabh Prakashan Publication, Delhi, Pg no. 295.

36.   N-alkylation of azoles disubstituted acetamides conditions by Ambarsingh P. Rajput and RambhauP.Gore, Der PharmaChemica, 2012

37.   Vinod. D. Rangari, Pharmacognosy and Phytochemistry vol 1, 3rd edition Pg no. 113-114.

38.   https://www.wikipedia.org/

39.   https://www.tandfonline.com

 

 

 

Received on 07.06.2019            Modified on 25.06.2019

Accepted on 18.07.2019            © A&V Publications All right reserved

Asian J. Res. Pharm. Sci. 2019; 9(3): 174-180.

DOI: 10.5958/2231-5659.2019.00027.4