INTRODUCTION:

Tuberculosis ^(1-3) is an infectious diseases caused by the bacillus Mycobacterium tuberculosis. It is the second leading cause of death from an infectious disease. The first line drugs involved in the treatment of TB are isoniazid, rifamycin, pyrazinamide, ethambutol and streptomycin. The second line treatment for TB are ethionamide, cyclocerin and capreomycin. But due to multi- drug resistance developed by the bacillus these drugs are proved to be losing efficacy and re occurrence of the disease is an many cases.

The mycobacterial cell wall, which is composed of mycolic acids (α-alkyl-β-hydroxy long chain fatty acids) is known to be important for the growth, survival, and pathogenicity of mycobacteria. Mycobacteria contain both type I (FAS I) and type II (FAS II) fatty acid biosynthetic pathways.

FAS is a single multifunctional polypeptide that catalyzes all the reactions in the elongation pathway. On the other hand, FAS II system is catalyzed by a series of small, soluble proteins that are each encoded by a discrete gene existing as separate proteins. Mycobacterium tuberculosis β-ketoacyl-acyl carrier protein synthase III (mtFabH) is a key condensing enzyme responsible for initiation of FAS II fatty acid biosynthetic pathway, and has emerged as an attractive new target for novel anti-tuberculosis agents in recent years. ^(4-5)

Pyrazolone derivatives are known to possess antitubercular, antifungal, anti-neoplastic activities.

Construction of our compounds containing both the pyrazolone and isonicotinic acid derivative systems towards the development of novel antimycobacterial agents. Based on we planned to link pyrazolone and isonicotinic acid derivative systems to produce better anti tubercular agents and to evaluate the interactions with the target(β-ketoacyl-acyl carrier protein synthase III ) by using ARGUS LAB docking software. ^(6-8)

MATERIALS AND METHODS:

MOLECULAR DOCKING^:(9-13)

Preparation of protein molecule:

The experimental structure of β-ketoacyl-acyl carrier protein synthase III (mtFabH) (PDB code-1HZP)) as shown in Figure 1 was retrieved from theRCSB protein data bank as a PDB file. The protein molecules were prepared mainly by using the software SwissPDB viewer. Active site residues within a range of 4.0 A0 were selected and saved in PDB format.

Preparation of ligand:

The ligand compounds pyrazolone derivatives were drawn using ACD/ Chemsketch (12.0) (Alex, 2009) andsaved in mol 2 format. The saved ligand compounds were later imported and minimized in Argus Lab after addinghydrogen bonds. The molecules thus obtained were saved in PDB format.

Argus Lab:

ArgusLab4.0 has fast become a favorite introductory molecular modeling package with academics mainly becauseof its user-friendly interface and intuitive calculation menus (Thompson, 2004). The ArgusDock docking engine,implemented in ArgusLab, approximates an exhaustive search methods. Flexible ligand docking is possible withArgusLab, where the ligand is described as a torsion tree and grids are constructed that overlay the binding site.Ligand’s root node (group of bonded atoms that do not have rotatable bonds) is placed on a search point in the binding site and a set of diverse and energetically favorable rotations is created. For each rotation, torsions inbreadth-first order are constructed and those poses that survive the torsion search are scored. The N-lowest energyposes are retained and the final set of poses undergoes coarse minimization, re-clustering and ranking

Docking of derivatives to β-ketoacyl-acyl carrier protein synthase III :

Docking of pyrazolonederivatives(INH I –INH V) with β-ketoacyl-acyl carrier protein synthase III (mtFabH) was performed using ARGUS LAB4.0. Thealgorithm exhaustively searches the entire rotational and translational space of the ligand with respect to thereceptors. The various solutions evaluated by a score, which is equivalent to the absolute value of the total energy of the ligand in the protein environment. The best docking solutions ARGUS LAB score for each compound wasconsidered. It was noted that ARGUS LAB scores of comp INH II and INH V was -8.68 and -8.86Kcal/molrespectively,which is greater than Isoniazid drug score value -6.17kcal/mol. as shown in Table 1, Figures 2,3& 4.

The drug like activity of the ligand molecules are characterized using ADME properties. Isoniazid andpyrazolone derivatives satisfy Lipinski rule of 5 and ADME properties results are shown in Table 2&3 .

Table 1. ARGUS LAB scores and interactions of isoniazid drug and pyrazolonederivatives

compound	Argus- Lab ( Kcal/mol)
INH I	-8.04
INH II	-8.68
INH III	-8.21
INH IV	-8.37
INH V	-8.86
Isoniazid	-6.17

Table 2&3.Lipinski rule of pyrazolone derivatives and isoniazid drug.

MOLECULAR PROPERTIES AND BIO ACTIVE SCORE OF COMPOUNDS

COMP	Log p	TPSA	MW	No. of hydrogen bond acceptor	No. of hydrogen bond conor	Violation	No. of rotable bond	Molar volume
INH I	1.182	103.76	385.81	8	2	0	5	321.98
INH II	0.463	149.58	396.36	10	2	1	6	331.77
INH III	0.561	112.995	381.39	9	2	0	6	333.99
INH IV	0.157	133.22	397.39	10	3	0	6	342.00
INH V	0.439	149.58	396.36	10	2	1	6	337.77

Comp	GPCR	Ion Channel	Kinase Inhibitor	Nuclear Receptor Ligand	Protease Inhibitor	Enzyme Inhibitor
	-0.20	-0.52	-0.20	-0.68	-0.43	-0.29
	-0.33	-0.53	-0.30	-0.71	-0.49	-0.34
	-0.23	-0.56	-0.21	-0.65	-0.43	-0.29
	-0.21	-0.52	-0.17	-0.59	-0.49	-0.29
	-0.33	-0.54	-0.30	-0.72	-0.50	-0.36SS

SYNTHESIS^(14-16)

STEP I:

A weighed quantity of Benzoic acid (substituted) 22.8gm (0.1mole) was dissolved in 50ml of ethanol in a RBF. Add 10ml of Con.H₂SO₄ to the reacting mixture and reflux for 1 ½ hr. The reaction mixture becomes concentrated by distillation. A crushed ice piece was added to the RBF and the solid mass becomes precipitate. It is filtered and dried.

STEP II:

Hydrazinolysis of benzoate(0.01 mol) with 10 ml of 99% hydrazine hydrate carried out with ester in presence of absolute ethanol (50ml) for 5-6 hr. After cooling and removing excess of solvent by distillation, the solid obtained was filtered, dried and recrystallised from ethanol.

STEP III:

A mixture of 0.01 mol of hydrazide and 0.1 mol(13ml) of ethyl acetoacetate were heated on water bath for 2hr with stirring from time to time with a glass rod. The resultant heavy reddish syrup was allowed to cool to room temperature. It was washed thoroughly with ether to remove coloured impurities. The solid separated out was filtered, dried and purified by recrystallization from ethanol.

STEP IV:

A mixture of 0.005mol (1.09gm) of 3-methyl pyrazol-5 one, 5ml of formaldehyde and 0.05mol (0.68gm) of isonicotinic acid hydrazide was refluxed with 25ml of 95% ethanol for 2hrs. The resultant mixture was concentrated. The resultant solid mass was dried and purified by recrystallization from ethanol.

PHYSICAL CHARACTERIZATION

GENERAL STRUCTURE:

TABLE NO: 4

Compound	R	Molecular Formula	Molecular Weight	Melting Point(⁰C)	Rf value
INH I		C₁₈H₁₆ClN₅O₃	385.80	162	0.71
INH II		C₁₈H₁₆ N₆O₅	396.35	130	0.32
INH III		C₁₉H₁₉N₅O₄	381.38	172	0.62
INH IV		C₁₉H₁₉ N₅O₅	397.38	256	0.47
INH V		C₁₈H₁₈N₆O₃	366.3	84	0.35

SPECTRAL STUDIES OF COMPOUNDS^:(17-18)

Compound INH I:

IR (KBr,cm^-1): 3150(CH(str) Aromatic) 1681.93 (C=O(str)amide) 1591(C=N(str) 761.88(C-Cl(str)) 3500(N-N(pyrrole)) 1425.4(C-H def (CH3)) 1062.78(C-N(str))

1485(C-H (def) (-CH2-)1307(C-C (str))

Compound INH II:

IR (KBr,cm^-1): 3342.64(CH(str) Aromatic ) 1670.35 (C=O(str)amide 1604.77(C=N(str)) 1521.84(Ar-NO₂ (N=Ostr))3413 (N-N(pyrrole))1062.78 (C-N(str)) 1440 (C-H (def) (-CH2-))

860.25(C-C (str). HNMR(DMSO-d6/TMS): (3H CH3)8.1, (8H C-H aryl2) 8.1- 8.3, (2H CH2) 4.19,(1H NH) 10.18

Compound INH V:

IR (KBr,cm^-1): 3219.19(CH(str) Aromatic) 1668.43 (C=O(str)amide) 1604.77(C=N(str)) 3400 (Ar-NH₂(C-Nstr)) 3327(N-N(pyrrole)) 1014.56 (C-N(str)) 1441.89(C-H (def) (-CH2-)) 840.96(C-C (str))

Anti-TB activity using Alamar Blue Dye: ^(19-21)

PROCEDURE:

1) The anti mycobacterial activity of compounds were assessed against M. tuberculosis using microplate Alamar Blue assay (MABA).

2) This methodology is non-toxic, uses a thermally stable reagent and shows good correlation with propotional and BACTEC radiometric method.

3) Briefly, 200µl of sterile deionzed water was added to all outer perimeter wells of sterile 96 wells plate to minimized evaporation of medium in the test wells during incubation.

4) The 96 wells plate received 100 µl of the Middlebrook 7H9 broth and serial dilution of compounds were made directly on plate.

5) The final drug concentrations tested were 100 to 0.8 µg/ml.

6) Plates were covered and sealed with parafilm and incubated at 37ºC for five days.

7) After this time, 25µl of freshly prepared 1:1 mixture of Almar Blue reagent and 10% tween 80 was added to the plate and incubated for 24 hrs.

8) A blue color in the well was interpreted as no bacterial growth, and pink color was scored as growth.

9) The MIC was defined as lowest drug concentration which prevented the color change from blue to pink.

RESULTS AND DISCUSSION:

The present study has given the development of new direct β-ketoacyl-acyl carrier protein synthase III (mtFabH) inhibitors. The virtual screening technique helped in stream lining promising (mtFabH) inhibitors from a vast library of compounds thus highlighting the potential of the lead moiety, pyrazolone linked with isonicotinic acid hydrazide as mtFabH inhibitor. The optimization of the lead obeyed the Lipinski’s rule of five and showed good drug likeness score predicting the leads to show good oral bioavailability.

Table no:5

S.NO	COMP	100	50	25	12.5	6.25	3.125	1.6	0.8
1	I	S	S	S	S	R	R	R	R
2	II	S	S	S	S	S	S	R	R
3	III	S	S	S	S	S	R	R	R
4	IV	S	S	S	S	S	R	R	R
5	V	S	S	S	S	S	S	S	R
6	ISONIAZIDE	S	S	S	S	S	S	S	S

Molecular docking has been utilized to conclude the relevance in synthesizing the leads. The synthesized compounds were characterized by IR and 1HNMR spectral data. The spectral datas confirms the successful formation of the newly synthesized compounds. All the compounds were obtained in good yields. The synthesized derivatives showed good anti tubercular activity confirming that they were promising candidates as mtFabH inhibitor. Compounds INH II and INH V showed excellent activity which is in correlation with the dock scores. These results highlight the identification of a new class of Anti mycobacterial agents that have potential to be more efficacious, than isoniazid, to treat tuberculosis.

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Received on 07.05.2014 Modified on 22.06.2014

Asian J. Research Chem. 7(7): July 2014; Page 681-686