Therefore, as part of our research effort on the elaboration of novel bio-active N and S heterocycles [ 33], as shown in Figure 1, we are very much engrossed in the design and efficient synthesis of the title compounds, which will be predictable to exhibit anti-bacterial activity due to co-existence of two kinds of pharmacophore entities. In this paper, we first described the one-pot synthesis and biological activity of a series of new thiazolyl-benzimidazoles (Figure 2). To our knowledge, not been reported so far.
Initially, to find the optimization conditions, we started our investigation to synthesize the title scaffolds starting from 5-amino-2-mercaptobenzimidazole (1), ammonium thiocyanate (2), and various substituted α-bromo acetophenones (3). To our delight, the desired benzimidazole-based thiazoles were obtained 70% of yield in the presence of glacial acetic acid at 70 ºC for about 4 h reflux (Table 1, entry-14) and its structure was unambiguously confirmed by IR, 1H-NMR, 13C-NMR spectra, and HRMS. Other solvents were also screened and it was revealed that DMSO, DMF, CH3CN, methanol, and ethanol resulted in very poor yields, while DMSO and CH3CN gave unsuccessful results (entries 1,2). Next, we examined the bases to improve the reaction yields, here we screened different organic and inorganic bases, but we did not observe greater yields when inorganic bases were used. Surprisingly, when fused sodium acetate was used as a base there is a sharp increase in reaction yield at 70 ºC within 4 h of time (Table 1, entry-15). But, when the reaction temperature increased a significant reduction in the product 4a yield was noticed (Table 1, entries-15,16). From this, we concluded that increasing the reaction temperature did not affect to improving the product yield (Table 1, entries-15,16). Further, different concentrations of the base were investigated. Finally, two equivalents of fused sodium acetate were found to be in the best condition with 88% yield (entry-15, Table-1).
Table 1. Optimized reaction conditions.4aa
Entry
|
Solvent
|
Catalyst
|
Temp (ºC)
|
Time (h)
|
Yield (%)
|
1
|
CH3CN
|
-
|
60
|
24
|
n.r
|
2
|
DMSO
|
-
|
60
|
24
|
n.r
|
3
|
DMF
|
-
|
60
|
20
|
21
|
4
|
Methanol
|
-
|
60
|
15
|
25
|
5
|
Ethanol
|
-
|
60
|
12
|
38
|
6
|
Ethanol
|
NaOH
|
60
|
12
|
43
|
7
|
Ethanol
|
KOH
|
60
|
12
|
45
|
8
|
Ethanol
|
Na2CO3
|
60
|
12
|
35
|
9
|
Ethanol
|
K2CO3
|
60
|
12
|
42
|
10
|
Ethanol
|
Et3N
|
60
|
12
|
41
|
11
|
Ethanol
|
Acetic acid
|
60
|
10
|
52
|
12
|
Ethanol
|
Acetic acid
|
60
|
6
|
60
|
13
|
AcOH
|
-
|
60
|
4
|
65
|
14
|
AcOH
|
AcONa (1.0 mmol)
|
60
|
4
|
70
|
15
|
AcOH
|
AcONa (2.0 mmol)
|
70
|
4
|
88
|
16
|
AcOH
|
-
|
reflux
|
4
|
51
|
aReaction conditions: 5-amino-2-marcaptobenzimidazole (1) (1.0 mmol), ammonium thiocyanate (2) (1.0 mmol), phenacyl bromide (3) (2.0 mmol), solvent (2 mL), bIsolated yields.
Under these optimized reaction conditions (Table 1, entry-15) the substrate scope of the reaction was studied using a series of α-bromo acetophenones. As shown in (Figure 2) different substituted α-bromo acetophenones either electron-withdrawing or electron-donating groups were well tolerated (4a-j) and presence of a strong electron-withdrawing group like nitro group on the phenyl ring offered the desired product 4i in excellent yield consequently presence of F, Cl, and Br groups on phenyl ring also (4b, 4c, 4d) offered products in good yield.
The final structure of the synthesized compounds (4a-q) was subjected to their spectral and analytical data. The 1H-NMR spectrum of compound 4a as a representative example showed a characteristic two singlet signals at 5.22 and 7.47 δ ppm due to -S-CH2 and C5-proton of thiazole respectively. The 13C-NMR spectrum displayed a significant signal at δC 193.06, 172.69, 107.89, and 21.50 δ ppm are assigned for C=O, C=N, C5 carbon of thiazole, and S-CH2 carbons respectively. The infrared spectra of compound 4a show frequencies at 3404 cm-1, 1685 cm-1, and 1623 cm-1 of amine (-NH), carbonyl (-C=O), and imine (C=N) functional groups respectively. The HRMS (ESI) spectra of all the synthesized compounds are shown [M+H] + as base peck.
Biological studies
Antibacterial results
The in-vitro antibacterial activity of synthesized thiazolyl-benzimidazole derivatives 4a, 4b, 4c, 4d, 4e, 4f, 4g, 4h, 4i, 4j, 4k, 4l, 4m, 4o, and 4p were analyzed against Gram-positive bacteria Streptococcus pneumonia (ATCC2451) and Gram-negative bacteria Proteus Mirabilis (ATCC2081). The in-vitro antibacterial activity of synthesized compounds was initially evaluated by determining their minimum inhibition concentration (MIC) values using the agar well diffusion method. Among the tested scaffolds, compound 4f has shown a maximum value of 3.6 cm of inhibitory activity against Gram Positive Streptococcus Pneumoniae (MTCC2451), and compound 4k has shown a value of 3.3 cm against Gram-negative Proteus Mirabilis (MTCC2081).
Table 2: Antibacterial activity of synthesized thiazolyl-benzimidazole scaffolds expressed as MIC against Proteus mirabilis (ATCC2081) and Streptococcus pneumoniae (ATCC2451).
|
Antibiotic activity by Zone of Inhibition (cm)
|
S.No
|
Compound
code
|
Proteus Mirabilis
(2081)
|
Streptococcus Pneumoniae
(2451)
|
1
|
4a
|
2.1+++
|
2.1+++
|
2
|
4b
|
2.25+++
|
2.85++++
|
3
|
4c
|
1.35++
|
2.1+++
|
4
|
4d
|
1.9++
|
1.8++
|
5
|
4e
|
2.75+++
|
2.55+++
|
6
|
4f
|
2.55+++
|
3.6++++
|
7
|
4g
|
1.45++
|
1.35++
|
8
|
4h
|
1.5++
|
2.1+++
|
9
|
4i
|
0.95+
|
1.2++
|
10
|
4j
|
1.9+++
|
2.25+++
|
11
|
4k
|
3.3++++
|
2.7+++
|
12
|
4l
|
2.3+++
|
2.25+++
|
13
|
4m
|
2.3+++
|
1.95+++
|
14
|
4o
|
2+++
|
2.25+++
|
15
|
4p
|
1.9+++
|
2.25+++
|
(++++ Maximum +++: Medium ++: Moderate + Minimum - Nil)
Structure-activity relationship (SAR)
The SAR studies were evaluated by changing the substituent on the 2,3,4 position of the phenacyl bromide ring as shown in Table 2 to see the impact of the electronic effects. Among the investigated compounds for their in-vitro antibacterial activity when the 4th position of phenacyl bromide ring was substituted with electron-withdrawing groups like F, Cl, Br, and nitro as in 4e, 4k showed excellent Gram-negative in-vitro antibacterial activity than compound with electron releasing or without any substituents. Further, compound 4f with electron releasing group (-OCH3) on phenacyl bromide ring was shown excellent Gram-positive in-vitro activity.
Molecular Docking and Molecular Dynamics Simulations
The calculated binding affinity values of the synthesized compounds at the active site of the gyrase B enzyme ranges from -9.8 to 12.0 kcal/mol. These values suggest that all the studied compounds show stronger affinity towards the active site of the receptor protein. Further, the difference in the docking score of two molecules is not very large which may be attributed due to the significant similarity in their chemical structure. The highest binding affinity value is observed for the compounds 4b, 4h, 4i, 4n, and 4p. The binding score is comparatively higher for the Phenyl (-Ph) substitution as in 4i and electron withdrawing substituents more particularly the nitro group (-NO2) as in 4h, 4n, and 4p.
The docked pose of the compound 4i along with 3D and 2D view of the interacting amino acids at the active site of the protein are shown in Figure 6. It is apparent from the figure that the binding pose of the studied scaffolds is unique and show a ‘dual-arm’ U-shaped binding mode on the protein. This U-shaped binding mode is uncommon in the most familiar gyrase inhibitors like quinolones and coumarins, however it was previously reported for kibdelomycin at the active site of Staphylococcus aureus GyrB. Although, U-shaped binding mode is observed at the active site of the GyrB, nevertheless, the binding position of the synthesized compounds substantially differs with that of the kibdelomycin. Therefore, the results exclusively emphasize that the synthesized compounds have the distinct binding mode at the active site of DNA gyrase B and may thus may be beneficial to inhibit the bacterial proliferation overcoming the cross-resistance for long practiced drugs.
Close analysis of the binding pose revealed that the substituted acetophenyl or benzyl moiety mostly occupies the ATP-binding pocket of the enzyme. The N and -NH of benzimidazole and N of thiazole ring usually establishes hydrogen bonding interaction with Asn45, Lys109, Val117 or Gly116 residue of the enzyme. The five-membered ring of benzimidazole or thiazole participates in the cationic-interaction with the Lys109 residue while the phenyl ring involves in the pi-stacking interaction with the Phe103 residue. Moreover, Asp45, Ile77, Phe103, Lys109, Val117, Pro328 are the amino acid residues which shows hydrophobic interactions with most of the synthesized molecules. The details of the binding affinity value and various binding interactions shown by the synthesized compounds with the protein along with the minimum distance from the residues are tabulated in Table 3 of Electronic Supporting Information (ESI).
En route to probe the reproducibility of the binding pose, the root mean square deviation (RMSD) between the two docked conformers have also been estimated at the active site of the receptor protein. The calculated RMSD value for the different compounds is also given in the Table 3 of ESI. It has been noticed that the RMSD values ranges between 0.581 to 2.361 which explains the reproducibility of the docking pose at the active site of the enzyme. Therefore, the studied compounds could potentially inhibit the functioning of DNA gyrase and serve as a promising antibiotic drug. Thereafter, an attention has been focussed to perform the molecular dynamics simulations for further insight into the stability of the compound at the active site of the gyrase protein. Figure 7 displays the plot of the variation in RMSD, radius of gyration (Rg), total energy, important distances with the amino acid residues during the simulation and the RMS fluctuation (RMSF) for the protein as well as the ligand.
It can be seen from the figure that the RMSD value of the gyrase protein progressively increased to 0.35 nm within 1.5 ns and stabilized in the range around 2.8–3.5 nm until the simulation time. Likewise, the RMSD value of the ligand is increased to 0.24 in the first 1.5 ns and fluctuates around 1.5–2.5 nm during the course of the simulation. Figure 7 also reveals that the Rg value only slightly increases from 2.46 to 2.53 nm in the initial 2 ns and then fluctuates within the range of 2.1–2.4 nm till the end of the simulations. Further analysis reveals that the total energy of the system almost remains the same with marginal fluctuations during the simulation. Interestingly, it has been observed that mostly RMSF of the residues in the protein more particularly the loop 98–118 (active site) is small. The RMSF value for most of the amino acid residues lies below 0.25 nm. Analogous to the protein, the fluctuations in each individual atom of the ligand are small and does not exceed beyond 0.2 nm. Moreover, the analysis of the variation in the distance unveils that the separation between the ligand and the amino acid residues Glu104, Gln105, Ala107, Tyr108, Lys109, Val110, Ser111, Asn271, Leu327, and Pro328 is maintained below 4 Å. All these evidences from the MD simulations advocates that the compound 4i is stable at the active site of the gyrase protein.
The geometries of the protein-ligand (4i) complex obtained at an interval of 2 ns are considered to elucidate the interaction of ligand (4i) at the active site of the receptor protein. The protein-ligand (4i) interaction in these complexes are unravelled using the PLIP and depicted in Figure 8. It is evident from the figure that the ligand molecule (4i) remains bound at the active site of the protein until the end of the simulation. Further, the unique U-shaped binding mode of the compound (4i) is retained during the entire simulations. Close analysis of the figure reveals that the interaction of 4i with Phe103, Lys109, and Pro328 is maintained throughout the simulations. The outcomes of the MD can be clearly visualized in a captured movie (movie.mpg) provided in the supporting information.