Novel 1 , 3 , 4-( thiadiazol-2-ylamino ) methyl-5-( pyridin-4-yl )-1 , 3 , 4-oxadiazol-2-thiones : synthesis , docking and antimycobacterial testing

In the present study, a novel series of Mannich bases of 3-substituted 5-(pyridin-4-yl)-1,3, 4-oxadiazol-2-thione derivatives were synthesized. Docking study was performed to rationalize the possible interactions between the synthesized compounds and active site of 14DM. The test compounds were screened for antimycobacterial activity using Middlebrook 7H9 medium against M. tuberculosis H37Rv (ATCC 27294) as well as Isoniazid (INH) resistant clinical strain. Among the series 5c and 5a are found to be most potent (susceptible) while the compound 5f did not show activity against M. tuberculosis H37Rv (resistant). The SAR study reveals the importance of substitutions at para position for good activity.


INTRODUCTION
Tuberculosis (TB) is a pandemic disease and its causative agent Mycobacterium tuberculosis is one of the most prolific infectious agents affecting humans.The 196 countries reporting to WHO in 2008 notified 5.6 million new and relapse cases in 2007, of which 2.6 million (46%) were new smear-positive cases [1].Furthermore, treatment of tuberculosis with human immuno deficiency virus infected patients (HIV) is difficult and results as the leading cause of death among HIV positive patients worldwide.Another factor which contributes to more number of deaths is the emergence of multiple drug resistance (MDR) [2][3][4][5].
Till now, no new drug has been introduced since the discovery of Rifampin in spite of major advances that have been made in the drug discovery process.Hence, there is an overwhelming need to develop novel antimycobacterial agents.Literature survey revealed that genomic DNA from the M. tuberculosis (MT) H37 Rvstrain, CYP 51-like gene encodes a bacterial sterol 14α-demethylase (MT P450 14DM), which acts on 14 α -methyl sterols.Recently we have reported a series of novel 4-(morpholin-4-yl)-N'-(arylidene) benzohy-drazides as antimycobacterial agent [19].In continuation of our project in development of new antimycobacterial agents, we thought of hybridizing the two potential pharmacophores i.e., 1,3,4-oxadiazole and thiadiazole which as such has been reported as antimycobacterial agents.Furthermore, solubility plays an important role for the development of drug in tuberculosis.Thus our aim was further refined to synthesize mannich base of these two pharmacophores and evaluate them for anti antimycobacterial activity.Herein we report the synthesis, docking and in vitro antimycobacterial activity of a series of mannich bases belonging to 5-(pyridine-4-yl)-1,3,4-oxadiazo l-2-thiones.The docking study was performed to rationalize the possible interactions between the synthesized compounds and the active site of 14DM.

Molecular Docking Protocol
All computations were carried out on a Wipro Intel Pentium processor with Windows XP Operating System.All the compounds were constructed using standard fragment library of Maestro 8.0 and geometry optimization was done by Macromodel program Schrödinger, LLC) using Optimized Potentials for Liquid Simulations-all atom (OPLS-AA) force field [23].The molecular docking tool, GLIDE (Schrodinger Inc., USA) was used for ligand docking studies into the X-ray crystal structure of cytochrome P450 14α-sterol Demethylase (14DM) from Mycobacterium in complex with 4-phenylimidazole (PDB entry code 1E9X) [24] were downloaded from the RCSB Protein Data Bank (PDB).The protein structure was prepared for docking using 'protein preparation wizard' in Maestro wizard 8.0.The protein preparation uses the OPLS force field 23 for this purpose.Grids were defined by centering them on the ligand in the crystal structure using the 10 Å box size.Ligprep 2.2 module utilized to produce the low energy conformer of ligands using MMFF94 force field [25].The lower energy conformations of the ligands were selected and were docked into the grid generated from protein structures using standard precision (SP) docking mode [26].

Docking and Scoring Functions
The docking studies were performed for the designed compounds with 14DM enzyme and the results were compared with the natural ligand phenyl imidazole present within the receptor.The docked complexes of the designed compounds along with the ligand receptor poses a composite Emodel score is then used to rank the poses of each ligand and to select the poses to be reported to the user.Emodel combines GlideScore, the nonbonded interaction energy, and, for flexible docking, the excess internal energy of the generated ligand conformation.

Antimycobacterial Activity
All the newly synthesized mannich bases were assayed in vitro for antitubercular activity against M. tuberculosis H37Rv (ATCC 27294) and Isoniazid resistant strain.The anti-TB screening was carried out by using Middlebrook 7H9 medium [27,28].

Modeling
The docked complex of the designed compounds is shown in Figure 1.The designed compounds were found to display good binding affinity to the receptor.

G-score, H-Bond Interaction and Contacts
The more negative value of G-score indicates that the compound is more potent and good binding affinity (Table 1).The Standard (CoCrystalisedLigand) and Isoniazid have shown G-Score of -4.73 and -5.02.The G-score of the designed compounds were found to be 5c > 5a > Isoni azid > 5b > 5e > standard co-crystallized ligand > 5d.
Besides the G-score, other parameters like energy and the E-model were also taken into consideration for the evaluation of the docking results; the values of the energy and E-model were found to be significantly more than that of the values of the standard co-crystallized ligand and Isoniazid.The designed compounds, 5a and 5d found to display 1 and 2 H-bonds with HIS 259 respectively.Standard (CoCrystalised Ligand) showed 1 H-Bond with HIS 259.It is well established and accepted fact that number of good van der Waals interactions decides the binding affinity for any ligand with receptor enzyme protein and bad, ugly contacts indicate steric clashes after docking which should be less for good activity.Therefore we have analyzed the binding  modes and abilities, considering the number of good, bad and ugly van der Waals (vdW) interactions of the standard and designed compounds with 14-DM active binding site.

Antimycobacterial Activity
Amongst the compound tested 5c and 5a have shown good antimycobacterial activity (susceptible) against M. tuberculosis H37Rv (ATCC 27294) among the series.The effect of other test compounds on H37Rv and Isoniazid resistant strain is given in Table 3.

DISCUSSION
The results obtained reveals that the nature of substituents on the aromatic ring have a considerable impact on the antitubercular activities of the test compounds.Literature survey indicates that electrons-withdrawing groups amend lipophilicity of the test compounds, which in turn alters permeability across the mycobacterial cell membrane.Furthermore, the presence of electronegative atom such as fluoro in 5c has shown better activity than 5e and 5b which have nitro at ortho and meta position respectively, clearly indicating the importance of para positions for substitutions.Also 5a bearing a methoxy group at para position have shown good activity.Nitro group in spite of being electron withdrawing does not show significant biological response, revealing the importance of para substitution than ortho and meta.Complete lose of activity in 5d and 5f observed may be due to bulky less and steric factors.This is well supported by the docking studies performed, as more the G score of the test compounds better the activity and binding ability of molecule into the active site.Our docking indicates good van der Waals interaction than the standard with 14DM.Unlike antifungal azoles, it had shown just a stacking effect with Fe of 14DM and not binding with Fe.

CONCLUSIONS
In conclusion a series of mannich bases containing two pharmacophores were synthesized and characterized.Molecular docking studies were performed in 14 DM protein docked with ligand to identify the possible inter-action.However two test compounds have shown better G score than Isoniazid.The test compounds were subjected to antimycobacterial study against H37Rv and INH Resistant Clinical Strain.Larger G score better the binding affinity of test molecules and is reflected in antimycobacterial activity indicating a direct correlation between observed activity and energy score.This indicates that the designed series of mannich bases possessing electron withdrawing group at para position have shown antimycobacterial activity.So, these factors collectively indicate the importance, simplicity and wide applicability of designed series as antimycobacterial activity.
A mixture of appropriate aromatic aldehyde 1 (1 g, 8.06 mmol), thiosemicarbazide (0.73g, 8.06 mmol) and catalytic amount of HCl in 10 ml ethanol were refluxed at 80˚C.The progress of the reaction was monitored by TLC.On completion, solvent were removed under vacuum.The solid obtained was recrystallized from 50% aqueous ethanol.
A mixture of thiosemicarbazone 2 (1g, 5.07 mmol) and ammonium ferric sulfate dodecahydrate (0.1 g) in H 2 O (5 ml) were refluxed for 1 h.Then 1 g ammonium ferric sulfate in H 2 O (10 ml) were added and further continued for 5 hr.On completion of reaction, the mixture was chilled; the solid separated was filtered off, washed and crystallized from EtOH-H 2 O.
Oxadiazole-2-thione 4 (0.5 g, 2.79 mmol) and substituted 2-amino thiadiazoles 3 (0.54 g, 2.79 mmol) were mixed in 15 ml absolute ethanol and stirred.To the stirred suspension (2.79 mmol, 37%) formaldehyde was added drop wise and heated to reflux for 10 -12 hrs.The progress of the reaction was monitored by TLC.On completion, reaction mixture was concentrated under reduced pressure and the residue obtained were recrysatllised from appropriate solvent.
Representative data from the series 3-((5-(4-methoxyphenyl)-1,3,4-thiadiazol-2-ylamino) methyl)-5-(pyridin-4-yl)-1,3,4-oxadiazole-2(3H)-thione The anti-TB screening was carried out by using Middlebrook 7H9 medium [25,26] against M. tuberculosis H37Rv (ATCC 27294).The basal medium was prepared, sterilized and to 4.5 ml of this broth, 0.5 ml of ADC (al-bumin-dextrose-catalase) supplement was added.Then a stock solution (10 mg/ml) of the test compounds was prepared and the final concentrations of 10, 25 and 50 µg/ml were transferred to media bottles.Finally, 10 µg suspension of M. tuberculosis H37Rv strain (100,000 organisms/ml adjusted by McFarland's turbidity standard) was transferred to each of the tubes and incubated at 37˚C along with one growth control without compound and drug control was also set up.The bottles were observed for growth twice a week for a period of three weeks.The appearance of turbidity was considered as growth and indicates resistance to the compound.The growth was confirmed by making a smear from each bottle and performing a Zeil-Nelson stain at the end of 4 weeks.

have been shown in the Figure 1 .
The final evaluation is done with glide score (docking score) and single best pose is generated as the output for particular ligand.: Van der Waal energy; Coul: Coulomb energy; Lipo: lipophilic contact term; HBond: hydrogen-bonding term; Metal: metal-binding term; BuryP: penalty for buried polar groups; RotB: penalty for freezing rotatable bonds; Site: polar interactions at the active site; and the Coefficients of vdW and Coul are: a = 0.065, b = 0.130.If GScore was selected as the scoring function,

Figure 1 .
Figure 1.(a) Binding mode of 5c inside the pocket of crystal structure 14 demethylase; (b) Binding of 5a in 14 demethylase.

Table 1 .
Results of molecular docking studies using standard precision mode of Glide.

Table 2 .
Prediction of ADME properties of designed derivatives using qikprop.Higher the value of MDCK cell, higher the cell permeability.All designed compounds have shown the ADME properties in acceptable range.