Synthesis, docking study and biological evaluation of some new thiourea derivatives bearing benzenesulfonamide moiety

Background A series of novel N-(2, 6-dimethoxypyrimidin-4-yl)-4-(3-(aryl)thioureido) benzenesulfonamides 3a–t was synthesized by the addition of N-(2,6-dimethoxypyrimidin-4-yl)-4-isothiocyanatobenzenesulfonamide 2 to the appropriate aromatic amine. The structures of the synthesized compounds were inspired from the second line antituberculosis pro-drugs. Results Most of the new compounds were screened for their activity against Mycobacterium tuberculosis. The results of the antimycobacterial assay showed that compound 3i exerted the highest activity (MIC = 3.13 µg/mL), followed by compound 3s (MIC = 6.25 µg/mL). Conclusion The structure–activity relationship (SAR) analysis revealed that the introduction of the benzo[1,3]dioxol moiety in 3i and the 4-morpholinyl-4-phenyl moiety in 3s has proven to give the most potent compounds in this study. Docking of the promising compounds inside the active site of M. tuberculosis enoyl reductase InhA was performed in order to emphasize the results. The compounds showed a similar orientation to that of GSK 625 inside the active site of 5JFO and bind to Met 98 in a way similar to that of the co-crystallized ligand.


Background
Tuberculosis (TB), is a disease caused by the facultative intracellular bacterium called Mycobacterium tuberculosis (MTB). WHO declared TB as a global health crisis [1] and a main cause of death due to the lack of appropriate treatment against resistant strains [2]. In 2012, TB was responsible for the death of 1.3 million people worldwide, Over 95% of them were from developing countries, also, TB represents the third cause of death for women aged 15-44. In addition, about one-third of the world's population harbors a dormant MTB infection, representing a significant incidence of the disease for the future [3]. TB treatment is tedious and time-consuming, that requires direct therapy and follow-up for not less than 6 months using these four drugs (isoniazid, rifampicin, pyrazinamide and ethambutol [1,4]. In addition, the recurrences of latent tuberculosis, are particularly prevalent in individuals with compromised immune system [5]. However, the present treatment protocols have proven to be underwhelming due to drug-drug interactions, intolerance, drug toxicity and poor patient adherence due to the lengthy treatment protocols [1,6]. That's why more effective and shorter treatment regimens are required. Thioureas act as precursors for the synthesis of different classes of acyclic and heterocyclic compounds, in addition to their high biological activity [7][8][9][10]. Second line antituberculosis pro-drugs as thioacetazone which is useful in preventing resistance to more powerful drugs such as isoniazid, isoxyl (thiocarlide) that is effective against multi-drug resistant strains, ethionamide (ETH) and prothionamide (Fig. 1) [11][12][13][14][15][16][17], were used to inspire the structures of our new thiourea derivatives, together with their mode of action. On the other hand, sulfonamides were largely employed as preventive and chemotherapeutic agents against various diseases [18], recent studies have shown that sulfonamides also possess antimycobacterial activity [19].
For the above-mentioned reasons and as a part of our interest in the synthesis and screening of potentially bioactive compounds [20][21][22][23][24], we herein, report the synthesis of some novel N-(2,6-dimethoxypyrimidin-4-yl)-4-(3-(aryl)thioureido)benzenesulfonamides 3a-t to be evaluated for their antimycobacterial activity. The promising compounds 3i and 3s were docked inside the active site of M. tuberculosis enoyl reductase InhA, to predict their possible mode of action. InhA enzyme was chosen as it contains a very hydrophobic site that favorably interacts with thioamide or thiourea moieties [25].

Chemistry
Isothiocyanates are widely used building blocks in the synthesis of nitrogen, sulfur and oxygen heterocycles [26]. The high electrophilicity and nucleophilicity associated with the carbon and sulfur atoms, respectively, of the isothiocyanates and their extended π electron system make them unique precursors for a large variety of target molecules. The intermediate, N-(2,6-dimethoxypyrimidin-4-yl)-4-isothiocyanatobenzenesulfonamide 2 [27] used for the preparation of the target compounds have been synthesized via thiophosgenation of sulfadimethoxine 1 at room temperature in the presence of dilute hydrochloric acid, according to the reported procedure (Scheme 1).
A series of N-(2,6-dimethoxypyrimidin-4-yl)-4-(3-(aryl) thioureido) benzenesulfonamides 3a-t was prepared by condensation of aromatic amines with N-(2,6-dimethoxypyrimidin-4-yl)-4-isothiocyanatobenzenesulfonamide 2 [27] in dioxane at reflux temperature in the presence of catalytic amounts of triethylamine, (Scheme 1). The structures of synthesized compounds 3a-t were confirmed by the absence of characteristic absorption band at 2000-2200/cm (N=C=S). Also, the IR of 3 is characterized by the presence of NH, thiocarbonyl (C=S) and SO 2 absorption bands. For example, the 1 H NMR spectrum of compound 3b showed two singlets at δ 3.81 and 3.84 ppm which were assigned for the two methoxy protons, a singlet at δ 6.1 ppm assigned to the pyrimidine-H, two downfield shifted singlets at δ 11.5 and 11.9 ppm which were readily assigned to the HN(1) and HN (2) protons, in addition to the presence of methyl, SO 2 NH and aromatic protons. The thiocarbonyl group of the thiourea moiety was also observed in the 13 C-NMR spectrum. The formation of thioureas 3a-t can be explained through the previously reported mechanism [24].

In vitro antimycobacterial activity evaluation
Evaluation of the synthesized compounds against M. tuberculosis (RCMB 010126) was initially carried out using the microplate Alamar blue assay (MABA) at the Regional Center for Mycology and Biotechnology (RCMB), Al-Azhar University (Cairo, Egypt) at a concentration of 200 µg/mL ( Table 1). As seen in Table 1, compound 3i was the most potent analog exhibiting good antimycobacterial activity that produced growth inhibition of 74.9%.
From the results in Table 2, it is apparent that the 4-position of the thiourea derivatives 3a-t, crucially affected the antimycobacterial activity. In which, incorporation of a Benzo [1,3]dioxol group in compound 3i led to good activity against M. tuberculosis (MIC = 3.13 µg/mL). The introduction of a methoxy group at 2-position of the spirodecane system increased the activity (except for 3b). The introduction of an electron-donating group at the 4-position, as methyl and methoxy groups, increased the activity. However, di-and trimethoxy substitutions (compounds 3c, 3d and 3g) led to decrease in the lipophilicity with a subsequent decrease in the antimycobacterial activity, indicating that the increased lipophilicity is crucial for the antitubercular activity.
It is well documented that increasing the lipophilicity, increases the diffusion through the lipid domain, thus, increasing the efficacy of the antimycobacterial agent [28][29][30][31].

Molecular docking
Tuberculosis is characterized by a number of drug targets namely: InhA, RpoB, DNA Gyrase, ATP synthase, and DprE1, inhibitors of those targets were found to be promising leads [32]. Isoniazid is still the most potent treatment targeting InhA enzyme. Isoniazid was found to interfere with Nicotinamide adenine dinucleotide (NAD)-utilizing enzymes, primarily the enoyl-ACP reductase encoded by the InhA gene, leading to the arrest of mycolic acid synthesis, which is essential to M. tuberculosis [32,33]. InhA enzyme was chosen based upon its hydrophobic properties that favorably interact with thioamide or thiourea moieties [25].
In our present study to determine the possible mode of action of the target compounds, molecular docking of compounds 3i and 3s was performed in the active site of Mycobacterium tuberculosis enoyl reductase InhA to explore their possible binding modes. The protein data bank file (PDB: 5JFO) was selected for this purpose. The file contains M. tuberculosis enoyl reductase InhA enzyme co-crystallized with [34]. All docking procedures were carried out using molecular operating environment (MOE) software 10.2008. Docking protocol was verified by re-docking of the co-crystallized ligand in the binding pocket of the enzyme with energy score (S) = −10.44 kcal/mol and root mean standard deviation (RMSD) = 0.39 (Fig. 2). The 2D ligand interaction of compound 3i (Fig. 3) demonstrates that the compound binds to the amino acid of the active site Met 98 through two hydrogen bonds (1.72, 2.44 Å).  Regarding compound 3s, the 2D and 3D ligand interaction simulations (Figs. 4 and 5) showed that 3s binds in the same fashion to the co-crystallized ligand displaying two hydrogen bonds with the active pocket amino acid Met 98 leading to an overall binding energy of = −11.64 kcal/mol (Table 3).

SAR (structure activity relationship)
From the results revealed by the antimycobacterial activity and the docking study, it is apparent that the group attached to the thiourea is crucial for the activity. The benzo [1,3]dioxol derivative 3i (MIC = 6.4 µM) was the most potent, followed by the 4-morpholinyl-4-phenyl derivative 3s (MIC = 11.8 µM), the oxygen atom of morpholine binds to Ser 20 inside the active site. Also, 3i and 3s have shown similar binding to that of the cocrystallized ligand inside the active site of M. tuberculosis enoyl reductase InhA and the best binding score in this series. The dipyridinyl- [1,2,4]triazole 3r and the 2-methyl-1,3-dioxo-2,3-dihydro-1H-isoindole derivative 3q also showed potent activity, with MIC = 21.7 and 43.5 µM, respectively. It is apparent that the nitrogens of the triazole ring in 3r tend to make additional binding interactions inside the active site of the enzyme as well as the carbonyl group in 3q, which may contribute to their antimycobacterial activity. C. Chemical shifts are expressed in δ values (ppm) relative to TMS as an internal standard, using DMSO-d6 as a solvent. Mass spectra were recorded on a 600 GC/MS (Clarus, Middletown, CT, USA) and TQ 320 GC/MS/MS mass spectrometers (Varian, West Sussex, UK). Elemental analyses were done on a model 2400 CHNSO analyzer (Perkin Elmer, Waltham, MA, USA). All reagents used were of the analytical grade.