science

1. http://www.rasayanjournal.com
Vol.4, No.4 (2011),868-874
ISSN: 0974-1496
CODEN: RJCABP
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: nalini_cn@yahoo.co.in
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 RASAYAN. 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).
NOVEL ISONIAZID DERIVATIVES C.N.Nalini et al.

2. Vol.4, No.4 (2011), 868-874
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.119°C: 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.122°C: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.128°C: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%) .
NOVEL ISONIAZID DERIVATIVES C.N.Nalini et al.
869

3. Vol.4, No.4 (2011), 868-874
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 37°C 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)
NOVEL ISONIAZID DERIVATIVES C.N.Nalini et al.
870

4. Vol.4, No.4 (2011), 868-874
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 ( μg/ml)
Microorganisms Standard
A1 A2 A3 A4 A5
NOVEL ISONIAZID DERIVATIVES C.N.Nalini et al.
871
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
300 300 350 400 300 12.3 15.2
aeruginosa
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
400 300 300 300 300 19.8 20.5
rubrum
Rhizopus nigricans 400 350 350 300 400 18.5 20.2
Sacchromyces
350 300 400 300 400 13.5 15.5
cereviasiae
Table-1(b)
Minimum inhibitory concentration ( μg/ml)
Microorganisms Standard
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

5. Vol.4, No.4 (2011), 868-874
NOVEL ISONIAZID DERIVATIVES C.N.Nalini et al.
872
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 Turkey’s 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

6. Vol.4, No.4 (2011), 868-874
100 66.6
150 83.3
200 100.0
O
R HN
Step (ii)
O
R HN
O
NOVEL ISONIAZID DERIVATIVES C.N.Nalini et al.
873
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
NH2
S
O O
Aldehyde/ketone
Isoniazid Schiff reaction
Sulphonamide
N
HN
O
N
R1
R2
N
N
O
N
R1
R2
HN
S
O O
N
Step (i)
Benzoylation
C6H5COCl
Step (iii)
Compound A1 to A5
Compound B1 to B10
R = -H, -Pyrimidine, -5-methyl isoxazole, -5,6-dimethoxy pyrimidine, -Pyridine
R1= -CH3 (B1-B4); -H (B5-B10);
R2= -H (B1B5); -p-bromo benzoyl (B2); p-hydroxy benzoyl (B3B9);-p-chloro benzoyl
(B4B7); -fluoro benzoyl(B6); -benzyl (B8); -p-nitro benzoyl (B10)
Scheme-1

7. Vol.4, No.4 (2011), 868-874
Table-3
Distance S.No. Docking Mode Affinity from best mode
(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
NOVEL ISONIAZID DERIVATIVES C.N.Nalini et al.
874
ISONIAZID
1 -4.4 0.0000 0.0000
REFERENCES
1. Sally-Ann Paulsen, Brendran L.Wilkinson, Caurent F.Bornaghi, Bio.org.med.chem.lett., 17,
1355(2007).
2. Mostafa M.Ghorab, Fatima A. Ragab, Mostafa M. Hamed , Eur.J.med.chem., 44, 4211(2000).
3. Subudhi B.,P.K Panda, Bhatta.D, J. Indian.chem.soc., 48B, 725(2009).
4. Ales Imramovsky, Slovenko Polanc, Jarmila Vinsova, Marijan Kocevar, Jampilek, Zuzuana Reckova,
Jarmila Kaustova, Bio.Org Med.Chem, 15, 2551( 2007).
5. Robert Thorn Morrison and Robert Neilson Boyd, Organic synthesis, 6th edition, 680, (2001)
6. Dharmarajan Sriram, Alexander Aubrey, Perumal Yogeswari, Fischer L.M., Bio.Org.Med.Chem.Lett,
16, 2982( 2006).
7. Grilles Klopmon, Sandhya Bawa, Suresh Kumar, J.Indian.Chem.Soc., 66, 252( 2001).
8. Yogesh Madhukar Nalavde, Vidya Joshi, Indian.J.Chem., 39B, 76( 2000).
9. Michael.J.Hearn, Michael.H.Gynamon, Michaeline.F.Chen, Rebecca Coppins, Jessica Davis,
Eur.J.Med.Chem, 44, 4189( 2009).
10. Tas Wang, Zhongxing Zhang, Nicholas.A.Meanwell, Tetra.Lett, 40, 6745( 1999).
11. OECD Guidelines for testing of chemicals, (1996).
12. Wilson and Grisvold, Textbook of Organic Medicinal and Pharmaceutical Chemistry, J.B.Lippincott
Company, USA, 9th edition, 387,( 1991).
13. Richard.A.Mark, R.Burns, Scott.A.Jenkins, Bio.Org.Med.Chem, 16, 1576(2006).
14. P. V. Ramana Rao, Essentials of microbiology. C.B.S. publication New Delhi 1st ed. ,8-9, 317( 2004).
15. R. S. Gaud, G. D. Gupta ,Practical microbiology. Nirali prakashan. Mumbai 3rd ed., 41( 2004).
16. P. R. Kumar and V.Vaidhyalingam , Der Pharm Lett., 2 (6), 16(2010)
17. H. Gerhard Vogel, Drug Discovery and Evaluation Pharmacological Assays, Springer-Verlag, Berlin
Heidelberg New York, 2, 670( 2002).
18. Rang and Dales, Rang and dales pharmacology, Elsevier publication London, 5th ed, 596-597(2004).
19. S.K Kulkarni, Hand Book of Experimental Pharmacology, Vallabh Prakashan., 3rd ed, 117-123( 1999).
20. William Foye, Principles of medicinal chemistry, Varghese Publication House Bombay, 2nd ed., 390-
398(1989).
[RJC-844/2011]