DESIGN AND SYNTHESIS OF NOVEL PYRIMIDINE ANALOGS AS ANTI-TUBERCULAR AGENTS TARGETING THYMIDINE KINASE DOMAIN

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INTRODUCTION
Tuberculosis is one of the deadliest diseases, which are caused by the bacteria, Mycobacterium tuberculosis (Dye et al., 1998;Dye et al., 2002).Globally, there were 9.2 million new cases and 1.7 million TB deaths in 2006, 0.7 million of which were in HIV-positive people and 0.2 million deaths (B.Greenwood et al., 2008).There are several flaws in existing drugs, the most prominent of which is the rise of drug resistance.For more than 20 years, no new medicines for tuberculosis have been discovered.Owing to the growing resistance to today's leading anti tubercular drugs, new therapies are urgently needed, which stimulated the pursuit of novel targets for the disease tuberculosis.The application of computational tools in the identification of drug targets, especially for Mtb, can very quickly produce a list of reliable targets.The inactivation of suitable novel targets, which are responsible for bacterial metabolism, growth and viability, would lead to bacterial death, thus eliminating the drug-resistant strains and shortening the duration of the therapy With this data, a study has been focused on screening some compounds containing pyrimidine moiety by using computational tools.And the compounds with good binding energy will be synthesized, and further will proceed for their antitubercular activity screening.

METHODOLOGY Materials and methods used
All the reagents were commercially obtained from Sigma Aldrich and utilized without further purification.Melting points were determined by capillary method and uncorrected.Shimadzu Perkin Ekmer 8201 Pc IR spectrometer (KBr Pellets), Bruker Avance II 400 NMR, JEOL SX-102/DA-6000 FAB Mass spectrometer were used for recording IR, NMR and Mass spectra.
The inhibition of the enzyme TMP kinase (TMPKmt), is hypothesized as a significant therapy for tuberculosis.A series of designed pyrimidines were synthesized to inhibit the enzyme TMPKmt and evaluated for their enzyme-ligand interactions, antitubercular, physiochemical and ADMET properties.The pyrimidines were synthesized from chalcones and guanidine as the cyclizing agent.The molecular interactions were studied by Autodock 4.0 and physicochemical, druglikeness and ADMET properties were analysed by Molinspiration, Chemsketch program and admetSAR prediction tools.The confirmation of the synthesized titled compound's structures was by spectral analysis.Also, they were screened for their antitubercular activity.In silico studies reports that their physicochemical, ADMET and druglikeness properties were found to be in standard limit, which infers that, these compounds may not have problems with oral bioavailability.Molecular docking studies showed that the pyrimidines have better enzyme inhibitory activity onTMPKmt.

Antitubercular activity
The method adopted for the antitubercular study was microplate Alamar blue assay (Collins et al.,1997) against M.tuberculosis.The diluted serial compounds (0.2 to 100.0 µM) and Middlebrook 7H9 broth (100 μl) containing Mycobacterium tuberculosis was poured to 96 well plates.The standard used was Isoniazid.After the sealing off the plates, incubation was done for seven days at 37ºC.After, the addition of the dye, it was re-incubated for 24h.The changes in the colour were noted, which was interpreted for pink as the growth of bacteria and blue as vice versa.

Software used
The software used for molecular docking is Autodock 4.0 (Goodsell et al., 1996) and the procedure followed for performing protein and ligand preparation is described elsewhere (Vanommeslaeghe et al., 2010;Morris et al., 1998;Chennu et al., 2015).Ligands were designed with the basic pharmacophore (pyridine) using pyrimidine as the primary nucleus with different cyclizing agents like guanidine and thiourea, substitutions like fluoro, chloro, bromo, nitro, methyl, methoxy, hydroxyl, and amino groups at 4 and 3 positions.About 12 compounds were designed for TMPKmt inhibition by incorporating these functional groups.Structure of the ligands was drawn using Chemsketch and SMILES notation was developed for the designed ligands.Molecular docking studies have carried out for compounds that have passed the ADMET (Feixiong et al., 2012) and Lipinski's rule of 5 (Lipinski, 2004).

Target and Ligand Preparation
TMPKmt crystal structure was downloaded from the Protein Data Bank with PDB ID as 4UNR (Naik et al. 2015).The repair commands module of AutoDock added the missing atoms.Before the docking process, the water molecules were removed, and H-atoms were supplemented to the targets, to for tautomerism and ionise amino acids.The modified structure was then applied for semi-flexible dockings.The energy minimization was performed by Discovery Studio (Version 4.0, Accelrys Software Inc 2007, with the CHARMM force field (Vanommeslaeghe et al.,2010).

Semi-flexible docking
The energy scoring grid box was customized to 126, 126 and 126 Å (x, y, and z) centered at X = 0.041; Y = -0.068and Z = 0.128 with 0.375 angstroms grid points spacing assigned with default atomic salvation parameters.Threedimensional grid boxes enclose the active site of the enzyme TMPKmt, which locates the ligand active binding site at the centre.Lamarckian Genetic Algorithm (LGA) (Morris et al., 1998) was the docking engine used in this study.After each LGA run, Autodock reports the best docking orientation, and the results were based on cluster analysis.The energy docking mode was found from a total of 10 docking modes.The lowest energy docking pose was chosen, from a total of 10 docking poses in each docking simulation.

Chemistry
Chalcone and guanidine in equimolar quantities with anhydrous potassium carbonate as the catalyst resulted in pyrimidines in better yields (Table 1).The IR spectra of compound PM1 shows an aromatic peak at 3415.8 and 3347.7 cm - 1 which corresponds to the amino group and C=N stretching at 1652 cm -1 .In addition, the 1 H NMR spectra predicted the presence of two hydrogens at 6.98 (s, 2H), which relates to aromatic NH2 group.Further, this compound got support from the mass spectrum, which depicted its molecular ion peak at 327, which corresponds to its molecular weight.

Antitubercular activity
The titled compounds were screened for their antitubercular activity by in vitro methods.All the compounds were found to have good antitubercular activity, and results are tabulated in Table 2. Compounds PM2 and PM4 were found to be active as standard, and their activity is due to the substitution of chloro and nitro in the 4-position of benzyl group of PM2 and PM4 respectively and the presence of the core groups pyridine and pyrimidine must have also contributed equally, with MIC value of 0.2 μM.With various other changes, an extensive structureactivity relationship could be derived in the future.

In silico studies
The predictions of drug-likeness and pharmacokinetic properties (ADMET) were performed by online tools molinspiration and admetSAR.The analogues owned desired physicochemical properties by satisfying all the Lipinski's RO5 properties with no violations from the standard limits (Table 3).The partition coefficient of all the compounds was found to be good values (1-3), which is essential for the absorption and distribution of drugs.ADMET properties were predicted, and it was found that all the parameters were within the acceptable range when compared with the standard (Table 4).Thus, all the hits were predicted with excellent physicochemical and druggable properties, rendering them good oral bioavailability.

Molecular docking
The designed compounds were found to have excellent binding affinity to the enzyme.The ten compounds were docked with TMPKmt domain protein to calculate its binding energy and understand its molecular interactions, which is in charge of target inhibition.Table 5, depicts the docking score of the docked compounds with the enzyme TMPKmt, within the range of -8.4 to -9.3Kcal/mol.Compounds PM3, PM7, PM8 obtained the best binding score (-9.3 Kcal/mol).This may be due to the great involvement of van der Waals and pi-pi interactions with the amino acids of the target.Figure 1a, describes the TMPKmt domain inhibited complex formation with the compound PM3 of maximum binding energy (-9.3 Kcal/mol), by forming pi-pi interaction with the residue Y103.Also showed van der Waals interactions with the residues L52; F70; S104; S99; Y103; R107. Figure 1b explains the TMPKmt domain inhibited complex formation with the compound PM4 of binding energy (-9 Kcal/mol), by forming two pi-pi interaction with the residue Y103.The complex also displayed two hydrogen bond of distance of 2.046 and 2.134 A and van der Waals relations with the amino acid residues Y103; R74; R95; N100; F70; S104; Y103; R107.The obtained interactions were found to be similar for the other docked pyrimidines, with the active amino acid residues, such as, Y103; R74; R95; N100; F70; S104; Y103; R107 and further, showed pi-pi interactions with the residues Y103.

CONCLUSION
In the present investigation, different pyrimidine derivatives were designed, synthesized and screened for their in silico properties such as physicochemical, druglikeness, and ADMET and the toxic-less compounds were docked and aligned to the active pockets of the TMPKmt enzyme.The results thus obtained revealed that the amino pyrimidines may have a considerable impact on the enzyme inhibitory activity.From these computational data, the molecular interactions between the ligand pyrimidines and the enzyme TMPKmt have been obtained.Thus, docking studies have proved that these pyrimidine derivatives, as ligands have a strong affinity for the enzyme TMPKmt, which might be a reason for its antitubercular activity.In conclusion, the investigated pyrimidines are found to be suitable lead structures with better inhibitory action towards the target TMPKmt.

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Zhang 2005; Mdluli et al., 2006).In the development of antitubercular drugs, thymidine monophosphate kinase of Mycobacterium tuberculosis (TMPKmt) (Gasse et al., 2008) is an obvious target.TMPK is a vital enzyme in the metabolism of Mycobacterium tuberculosis, and also, it is the precise enzyme in dTTP synthesis.It is unlike human enzyme analogues (22% homology).It catalyses the conversion of deoxythymidine monophosphate (dTMP) to deoxythymidine diphosphate (dTDP) using ATP as a phosphoryl donor (Lavie et al., 1998; Ostermann et al., 2001).Due to its prospects as antimetabolites, TMPK inhibitors have gained considerable interest.Even, by inhibiting DNA synthesis in M. tuberculosis, these molecules can be promising leads in treating tuberculosis.Very few studies of lead generation and optimisation have been explored for TMPKmt inhibitors (Pochet et al., 2003; Pochet et al., 2010).Pyrimidines have been investigated extensively among the plethora of nitrogencontaining heterocycles.This chemical moiety is of paramount importance due to its activities such as antitubercular (Breda et al., 2012), anticancer (El-Deeb et al., 2010), (Tan et al., 2014), antimalarial (Singh et al., 2013), antitumor (El-Nassan et al., 2011), analgesic, anti-convulsant (Deng et al.,2011) etc.Since only limited reports are available about pyrimidines as anti-tubercular agents, hence, the design of novel pyrimidines with its target is a feasible choice (Singh et al., 2011).

Figure 1a )
Figure 1a) 3D representation of the molecular interactions of compound PM3, within the binding pocket of protein and forming a pi-pi interactions with Y103 residue

Table 1
Physical data of synthesized pyrimidines

Table 2
Antitubercular activity of synthesized pyrimidines

Table 3
In silico physicochemical properties of synthesized pyrimidines

Table 4
In silico ADMET studies of pyrimidines

Table 5
Docking results of the pyrimidines with TMPKmt receptor domain