nalini, 2011

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Vol.4, No.4 (2011),868-874 ISSN: 0974-1496

CODEN: RJCABP

NOVEL ISONIAZID DERIVATIVES C.N.Nalini et al.

STRUCTURE BASED DRUG DESIGN, SYNTHESIS, CHARACTERISATION AND BIOLOGICAL EVALUATION OF

NOVEL ISONIAZID DERIVATIVES

C.N.Nalini*1, Arivukkarasi2 and R. Devi2 1Department of Pharmaceutical Chemistry, C.L.Baid Metha College of Pharmacy, Chennai-97.

2Department of Pharmaceutical Chemistry, C.L.Baid Metha College of Pharmacy, Chennai-97. *E-mail: [email protected]

ABSTRACT Some Novel biologically active Isoniazid derivatives substituted with Sulphonamides and Aldehydes were synthesized and the same were characterized by IR, 1H-NMR, and MASS spectral analysis. The compounds were subjected to antimicrobial screening (both in vitro and in vivo) and the results are compared with the standards. Insilico screening was done with AUTODOCK 4 which is a suite of automated docking tools. All the synthesized compounds were docked with DNA GYRASE (TopoisomeraseII type). The docking study data showed best fit Root Mean Square Difference (RMSD). The in-vivo antibacterial evaluation revealed that the 2-bromo-1-phenylethylidene and 4-fluorobenzylidene substituted isoniazid derivatives showed very potent activity. Keywords: Isoniazid, Sulphonamides, Aldehydes, DNA Gyrase, Antimicrobial.

2011 RASYAN. All rights reserved.

INTRODUCTION Sulphonamides were the first antimicrobial drugs and paved the way for Antibiotic revolution in medicine. Isoniazid is the first line antitubercular medication used in the treatment and prevention of Tuberculosis. In this modern world the drug discovery involves medicinal chemistry along with other important fields like CADD (Computer Aided Drug Discovery), 3D QSAR, X-Ray crystallography and pharmacokinetic studies etc,. Docking is one of the computational methods to know about the ligand receptor protein) interactions. Present work includes structure based design, synthesis and biological evaluation of some novel Isoniazid4,5 derivatives with Sulphonamides1-3 and aldehydes (SCHIFF REACTION) followed by benzoylation of the Schiff bases6. Insilico screening was done by Auto docking the target enzyme DNA Gyrase7,8 (Topoisomerase II type) using AUTODOCK software. Novel Isoniazid derivatives were characterized by spectral methods like IR, 1H-NMR and Mass spectra. Synthesized compounds were screened for antimicrobial and antitubercular activities and compared with known standards.

EXPERIMENTAL General procedure for the synthesis of 4-substituted Isonicotinamides (A1-A5) To sulphonamides (0.003mol) in dry benzene (15ml) was added to Isoniazid (0.003mol) in dry benzene (15ml) and the reaction mixture was refluxed for 3 hours. The product9 was then cooled, filtered and dried. The procedure was repeated with different sulphonamides. General procedure for the synthesis of Schiff bases of Isoniazid A mixture of equimolar quantities of aldehydes and Isoniazid 10 were taken in an beaker. To this 10 ml of ethanol and few drops of glacial acetic acid were added and kept in reflux for 1 to 3 hours. The reaction mixture was poured on crushed ice or ice cold water. The separated product was filtered out, washed and recrystallised from ethanol. The ten different Schiff bases were obtained similarly by changing the calculated quantity of different aldehyde with respective to mole ratio. Schiff bases obtained from the above reaction were used for the synthesis of final compounds (B1-B10).

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General procedure for the synthesis of title compounds: (B1-B10) The schiff bases and aqueous sodium hydroxide were mixed in 250 ml conical flask fitted with a rubber cork and shaken well. Benzoyl chloride was added slowly as in 1ml increment and shaken well for 15-20 mins. The disappearance of aroma of benzoyl chloride and appearance of brownish solid indicated the completion of reaction. At once the reaction ended the flask cooled and filtered the product11. Washed and dried well. Recrystallised form hot methylated spirit. Equipment Melting points of the synthesized compounds were determined in open capillary tubes and uncorrected. The IR spectroscopy was performed by ABB Bomen FTIR Spectrometer MB 104 using KBr pellets. The 1H-NMR spectra of synthesized compounds were recorded on a JOEL GSX 400 NMR Spectrophotometer in DMSO. The MASS Spectra was recorded on GCMS QP-5000 SHIMADZU. The homogenecity of all the newly synthesized compounds were routinely checked by Thin Layer Chromatography on silica gel (HF254 200 mesh) using chloroform:methanol as eluent and visualized in UV chamber.

The spectral data of all the title compounds (B1-B10) N-benzoyl-N-(1-phenylethylidene) Isonicotinohydrazide (B1) Mol.formula-C12H17N3O2 . Yield :63%, m.pt.119C: IR(KBr,cm-1): 3071(C-H aromatic str), 1583 (C=C str),1599(C=O str), 1583(C=N str),1213,1175(30 NH3).1 H NMR (ppm): 7.3-8.8(m)-CH-aromatic, 2.5 (CH-). MS: Mol.wt 343.15, 342(M+ ) (8%) . N-benzoyl-N-(2-bromo-1-phenylethylidene)Isonicotinohydrazide (B2) Mol.formula-C21H16BrN3O3. Yield:62%, m.pt.122C:IR(KBr,cm-1): 3073(C-H aromatic str), 1602 (C=C str), 1705(C=O str), 1583(C=N str), 1271,1177(30NH3),. 1H-NMR( ppm): 6.7-9.3 (m) CH- aromatic, 2.7 (-CH2-). MS: Mol.wt 421.04,420(M+ ) (8%) . N-benzoyl-N-(2-hydroxy-1-phenylethylidene)Isonicotinohydrazide (B3) Mol.formula-C21H17N3O3. Yield:78%, m.pt.128C:IR(KBr,cm-1): 3071(C-H aromatic str), 1601 (C=C str), 1686(C=O str), 1588(C=N str), 1292,1175(30NH3), 3382(C-OH). 1H-NMR( ppm): 7.2-8.3 (m) CH- aromatic, 3.3 (-CH2-),2.5((s)-OH). MS: Mol.wt-359.13 , 359(M+ ) (14%) . N-benzoyl-N-(2-chloro-1-phenylethylidene)Isonicotinohydrazide (B4) Mol.formula- C21H17ClN3O3. Yield:66%, m.pt.1340C:IR(KBr,cm-1): 3072(C-H aromatic str), 1599 (C=C str), 1721(C=O str), 1583(C=N str), 1213,1175(30NH3), 3382(C-OH). 1H-NMR( ppm): 6.3-9.5 (m) CH- aromatic, 2.3 (-CH2-). MS: Mol.wt-377.09 , 377(M+ ) (8%) . N-benzoyl-N-methylene-Isonicotinohydrazide (B5): Mol.formula- C14H11N3O3. Yield:64%, m.pt.1230C:IR(KBr,cm-1): 3071(C-H aromatic str), 1601 (C=C str), 1686(C=O str), 1583(C=N str), 1292,1175(30NH3). 1H-NMR( ppm): 7.5-9.5 (m) CH- aromatic,11.0 (-CH2-). MS: Mol.wt-253.07 , 252(M+ ) (42%) . N-benzoyl-N-(4-fluorobenzylidene)-Isonicotinohydrazide (B6) Mol.formula- C20H14FN3O2. Yield:61%, m.pt.1240C:IR(KBr,cm-1): 3071(C-H aromatic str), 1599 (C=C str), 1721(C=O str), 1583(C=N str), 1325(C-F) 1213,1175(30NH3). 1H-NMR( ppm): 6.3-8.9 (m) CH- aromatic,9.3 (-CH-). MS: Mol.wt-347.11 , 347(M+ ) (10%) . N-benzoyl-N-(4-chlorobenzylidene)-Isonicotinohydrazide (B7) Mol.formula- C20H14ClN3O2. Yield:71%, m.pt.1190C:IR(KBr,cm-1): 3072(C-H aromatic str), 1600 (C=C str), 1692(C=O str), 1583(C=N str), 1291,1275(30NH3),706(C-Cl str). 1H-NMR( ppm): 7.2-8.9 (m) CH- aromatic,9.3 (-CH-). MS: Mol.wt-363.08 , 363(M+ ) (9%) . N-benzoyl-N-benzylidene-Isonicotinohydrazide (B8) Mol.formula- C20H14N3O2. Yield:65%, m.pt.1260C:IR(KBr,cm-1): 3081(C-H aromatic str), 1602 (C=C str), 1688(C=O str), 1584(C=N str), 1292,1172(30NH3). 1H-NMR( ppm): 7.2-8.3 (m) CH- aromatic,9.3 (-CH-). MS: Mol.wt-329.12 , 329(M+ ) (42%) . N-benzoyl-N-(4-hydroxybenzylidene)-Isonicotinohydrazide (B9) Mol.formula- C20H15N3O3. Yield:72%, m.pt.1350C:IR(KBr,cm-1): 3071(C-H aromatic str), 1600 (C=C str), 1688(C=O str), 1583(C=N str), 1291,1178(30NH3),3382(C-OH). 1H-NMR( ppm): 6.0-8.9 (m) CH- aromatic,9.3 (-CH-), (s)-OH-5.2. MS: Mol.wt-345.10 , 345(M+ ) (19%) .

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N-benzoyl-N-(4-nitrobenzylidene)-Isonicotinohydrazide (B10) Mol.formula- C20H14N4O4. Yield:67%, m.pt.1310C:IR(KBr,cm-1): 3071(C-H aromatic str), 1602 (C=C str), 1584(C=N str), 1292,1172(30NH3), 1531(C-NO2). 1H-NMR( ppm): 7.2-8.8 (m) CH- aromatic,8.8 (-CH-). MS: Mol.wt-374.10 , 374(M+ ) (20%) . Acute oral toxicity tests Acute oral toxicity studies of the synthesized compounds were performed by acute toxic class method and were performed according to the OECD guideline 423 method 12. From the toxicity study the compounds were proved to be non-toxic at tested dose level (2000mg/kg) and well tolerated by experimental animals. Insilico Screening Docking can be applied to computationally screen a given compound against a specific target protein. Computational method that predicts the 3D structure of a protein-ligand complex is the objective of molecular docking approaches. Protein structure can be employed to dock ligands into the binding site of the protein and to study their interactions. The Protein Data Base (PDB) currently holds more than 20,000 protein crystal structures. Auto Dock 4 is a suite of automated docking tools. A binding interaction between a small molecule (ligand) and an enzyme (protein) may result in activation or inhibition of the enzyme. Here the target (protein) enzyme of our interest is DNA Gyrase which is an enzyme that unwinds double stranded DNA. DNA Gyrase is a type II-topoisomerase which introduces negative super coils into DNA by looping the template so as to form crossing then cutting one of the double helices. Molecular docking study was employed for the analysis with training set of 15 compounds. Standard isoniazid was also docked for comparative studies. All the derivative of Isoniazid showed best fit Root Mean Square Difference (RMSD) values of 0.0000. The PDB id of DNA Gyrase is 3ILW protein. Among all compounds B10 (with p-nitrobenzylidene substituted) showed high affinity and low energy of -6.2 kcal/mol and compared with STD Isoniazid which showed low energy value of -4.4 kcal/mol with RMSD value of 0.0000.

Biological Evaluation Anti Microbial Tests(In-vitro method) Of various methods available to evaluate the antimicrobial activity the cup-plate method was used to do in-vitro antimicrobial screening of synthesized compounds Mirco organisms used are- Gram +ve (Mycobacterium phlei, Bacillus cereus, Staphylococcus aureus, Bacillus subtilis) Gram ve (Escherichia Coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Salmonella typhii) Fungi (Candida albicans, Aspergillus niger, Trichophyton rubrum, Rhizopus nigricans, Sacchromyces cereviasiae). Medium used: (Nutrient agar medium and Sabouraud dextrose agar medium)Standard drugs: Ciprofloxacin (anti bacterial); Ketoconazole (anti fungal). In-vivo Antibacterial activity The in-vivo activity of all the synthesized compounds against systemic infections in male Swiss albino mice was determined by mouse protection test. Four week old male Swiss albino mice weighing 18 to 22g were used for systemic infection model. Test organisms for infections were cultured in Hinton nutrient agar medium at 37C for 18 hrs. For use as inoculate, E.coli was suspended in 0.9% saline solution containing 5% gastric mucin. Mice were used in groups of six for each inoculum and were challenged intraperitonially with a single 0.5 ml portion of the bacterial suspension. Various dose regimens like 50,100,150,200mg/kg of synthesized compounds were orally administered to mice twice at 1 and 4th hr of post infection. Synthesized compounds were suspended in 1% Carboxy Methyl Cellulose. Mortality was recorded 7 days, and the median effective dose needed to protect 50% of the mice (ED50) was calculated by intrapolation among survival mice (% protection) in each group after a week.

RESULTS AND DISCUSSION The N-substituted Isoniazid derivatives were subjected to insilico screening (docking studies). The docking study data revealed that all the compounds showed best fit (RMSD) value with topoisomerase (DNA Gyrase). All the compounds (A1 to A5, B1 to B10) were synthesized according to the synthetic scheme and characterized by IR, 1H-NMR and mass spectroscopy. Compounds (A1 to A5, B1 to B10)

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were subjected to in-vitro antimicrobial screening by cup plate method and the MIC was evaluated by test tube dilution method. The compounds (A1 to A5, B1 to B10) were subjected to acute oral toxicity study as per OECD guidelines 423 method. Compounds were evaluated for in-vivo antimicrobial activity against E.coli.

Table-1(a)

Minimum inhibitory concentration ( g/ml) Standard Microorganisms

A1 A2 A3 A4 A5 25 50

Gram Positive Mycobacterium phlei

300 400 400 300 300 6.4 7.5

Bacillus cereus 300 300 350 400 400 7.0 8.9 Staphylococus aureus

300 300 350 400 300 11.3 12.1

Bacillus subtilis 300 300 400 300 400 11.2 13.5 Gram Negative Eschericia coli 350 300 300 500 300 15.4 20 Pseudomonas aeruginosa

300 300 350 400 300 12.3 15.2

Klebsiella pneumonia

400 300 400 400 400 14.2 16.1

Salmonella typhi 300 300 400 300 350 12.9 14.4 Fungi Candida albicans 300 300 300 300 300 19.7 21.3 Aspergillus niger 400 350 300 300 350 15.4 16.3 Trichophyton rubrum

400 300 300 300 300 19.8 20.5

Rhizopus nigricans 400 350 350 300 400 18.5 20.2 Sacchromyces cereviasiae

350 300 400 300 400 13.5 15.5

Table-1(b)

Minimum inhibitory concentration ( g/ml) Standard Microorganisms

B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 25 50

Gram Positive Mycobacterium phlei 350 150 300 150 400 100 100 300 300 150 6.4 7.5

Bacillus cereus 350 150 350 100 300 150 150 300 350 100 7.0 8.9 Staphylococus aureus

500 100 500 100 400 100 100 300 300 150 11.3 12.1

Bacillus subtilis 500 150 500 100 300 50 150 400 400 100 11.2 13.5 Gram Negative Eschericia coli 300 50 300 150 350 100 150 400 300 150 15.4 20.0 Pseudomonas 300 100 350 100 350 100 100 400 300 100 12.3 15.2

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aeruginosa Klebsiella pneumonia 300 100 350 150 400 100 100 400 300 150 14.2 16.1

Salmonella typhi 300 100 400 100 300 100 150 400 350 100 12.9 14.4 Fungi Candida albicans 400 100 300 150 300 100 100 300 400 100 19.7 21.3 Aspergillus niger 400 100 300 100 400 50 150 300 350 50 15.4 16.3 Trichophyton rubrum 400 100 300 100 300 150 100 300 300 100 19.8 20.5

Rhizopus nigricans 400 100 350 50 300 150 100 400 300 50 18.5 20.2 Sacchromyces cereviasiae 350 150 400 100 300 50 100 350 300 100 13.5 15.5

Standards used: Ciprofloxacin Antibacterial; Ketoconazole Antifungal

The compounds containing bromo and fluoro substitution have exhibited significant antimicrobial activity. The compounds (A1 to A5, B1 to B10) represent a fruitful matrix for the development of a new class of compounds that would deserve further investigation and derivatisation. Table -3 furnishes details of docking studies. In-vitro antimicrobial activity The cultures were incubated at a temperature optimal for growth of test organisms (usually 24 hrs for bacteria at 370C and 48 hrs for fungi at 270C). Among the compounds evaluated for antibacterial activity, few compounds possessed reasonably good activity against gram positive and gram negative bacteria. All the compounds showed moderate activity when compared with that of standard Ciprofloxacin. When the synthesized compounds were tested for their antifungal activity, few compounds showed moderate activity against Candida albicans, Aspergillus niger, Trichophyton robrum, Rhizopus nigricans, Sacharomyces cereviasiae when compared with that of the standard Ketoconazole.The minimum inhibitory concentration (MIC) of the compounds against various bacteria and fungi were listed on Table-1 (a) and (b). In-vivo anti bacterial activity Among the synthesized compounds evaluated B2 and B6 showed very potent activity, compounds B7, B10 showed equipotent activity and compound B4 showed significant activity compared with Ciprofloxacin.Table 2 furnishes the percentage protection and ED50 values of the compounds evaluated against systemic infection by E.coli. Statistical analysis The statistical analysis was carried out by one way ANOVA (Analysis of Variance) method. The values are represented as mean S.E.M. Comparison of mean values of different groups treated with different dose levels of formulation and positive control with normal were estimated by Turkeys multiple comparison tests.

Table-(2): Invivo data of the synthesized compounds against E.Coil

S.No. Compounds Dose Percentage Protection(%) ED50

mg/kg 50 33.3 100 66.6 150 83.3 1. B2

200 100.0

75.0

50 66.6 100 33.3 150 83.3 2. B4

200 50.0

200.0

3. B6 50 33.3

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100 66.6 150 83.3 200 100.0

75

50 33.3 100 50.0 150 66.6 4. B7

200 100.0

100

50 66.6 100 33.3 150 50.0 5. B10

200 100.0

150

N

HN

O

NH2

R1 R2

O

NH2

SOO

HNR

Aldehyde/ketoneSchiff reactionIsoniazid

Sulphonamide

N

HN

O

N

R1

R2

Step (ii)

N

N

O

N

R1

R2

O

HN

SOO

HNR

O

N

Step (i)BenzoylationC6H5COClStep (iii)

Compound A1 to A5Compound B1 to B10

R = -H, -Pyrimidine, -5-methyl isoxazole, -5,6-dimethoxy pyrimidine, -PyridineR1= -CH3 (B1-B4); -H (B5-B10); R2= -H (B1&B5); -p-bromo benzoyl (B2); p-hydroxy benzoyl (B3&B9);-p-chloro benzoyl (B4&B7); -fluoro benzoyl(B6); -benzyl (B8); -p-nitro benzoyl (B10)

Scheme-1

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Table-3

Distance from best mode S.No. Docking Mode Affinity (Kcal/mol) RMSD i. b RMSD u. b

01 3ILW Vs A1 1 -6.1 0.0000 0.0000 02 3ILW Vs A2 1 -5.6 0.0000 0.0000 03 3ILW Vs A3 1 -5.8 0.0000 0.0000 04 3ILW Vs A4 1 -5.6 0.0000 0.0000 05 3ILW Vs A5 1 -5.9 0.0000 0.0000 06 3ILW Vs B1 1 -5.7 0.0000 0.0000 07 3ILW Vs B2 1 -5.4 0.0000 0.0000 08 3ILW Vs B3 1 -5.3 0.0000 0.0000 09 3ILW Vs B4 1 -4.9 0.0000 0.0000 10 3ILW Vs B5 1 -5.4 0.0000 0.0000 11 3ILW Vs B6 1 -6.1 0.0000 0.0000 12 3ILW Vs B7 1 -6.1 0.0000 0.0000 13 3ILW Vs B8 1 -6.1 0.0000 0.0000 14 3ILW Vs B9 1 -6.0 0.0000 0.0000 15 3ILW Vs B10 1 -6.2 0.0000 0.0000 16 3ILW Vs

ISONIAZID 1 -4.4 0.0000 0.0000

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