3.1. Chemistry:
Among the derivatives of 2,3-diaminopyridine, 5-bromo-2,3-diaminopyridine appears to be a potentially important synthon involved in the synthesis of 6-Bromo-2-phenyl-1,3,4-triazaindan [31]. The condensation of this compound with benzaldehyde led to the expected formation of the 1,3,4-triazaindan derivative (1, Fig. 2):
Figure 2
Conventional alkylation techniques which involve weak bases or strong bases have a number of disadvantages: They are expensive, very slow, and lead to the formation of byproducts which are difficult to purify. To circumvent these challenges, we developed another highly efficient alkylation method: phase transfer catalysis (PTC) using K2CO3 as the base and t-BAB as the catalyst [32–33]. We continued our experiments in applying this method for the preparation and study of the antimicrobial activity 1,3,4-triazaindan derivatives. The tautomeric form present in the 1,3,4-triazaindan skeletons (1) made this system more diversified, and the condensation of 6-Bromo-2-phenyl-1,3,4-triazaindan with 1.2 equivalents of alkyl halides under (PTC) conditions affected two positions: the nitrogen at position 3 (N3) and at position 4 (N4) (Fig. 3):
Figure 3
Further, the action of bis(2-chloroethyl)amine hydrochloride under phase transfer catalysis in the presence of an excess of potassium carbonate enabled the formation of an isoxazolidine ring with a satisfactory overall yield (Fig. 4):
Figure 4
All the obtained compounds were purified via column chromatography and isolated in good overall yields (80–91%, Table 1). The molecular structures of the new compounds were established on the basis of the 1H and 13C NMR spectroscopic data and mass spectrometry.
3.2. Crystallographic data
A crystallographic study performed on compound (3a) showed that alkylation took place in the 3rd position (Figure S.1, Table S.1, Supplementary Materials) [34].
3.3. Hirschfield profile for Molecular Packing 3a
The intermolecular interactions and Hirschfield-surfaces "HF" that contribute to the stability of the 3a molecular packing are shown in Fig. 5. The dnorm was computed as [35], where “rvdWi”and”rvdWe” are based on the closest interaction between internal and external particle morphology using VanderWaals-radii. The"+" dnorm value for short rvdW, and a "−" value for long rvdW. The relationship between the "de"and"di" of the HF for 3a was obtained with the crystal-explorer [36]. The 3D-HF of the 3a molecule is depicted in (Fig. 5) and has(-0.166Åto1.4059Å) for dnorm; (1.0370Åto2.5919Å)for di; (1.0566Å to 2.6244 Å) for de; (-1.00 Å to 1.00) for shape index; (-4.00Å to 4.00Å) for curvedness; and (0.00Å -13.00Å) for patch fragmentation. The dnorm fingerprint shows the hydrogen contacts as the red area, which were longer than the vdWs-radii. The shortest interactions was arranged by as Br•••N/N•••Br (1.5%) and Br•••C/C•••Br (1.1%) to packing molecule. The H•••C/C•••H, O•••H/ H•••O, and Br•••H/H•••Br interactions exceeded the vdWs radii by 10.7%, 14.1%, and 15.6%, respectively. The N•••H/H•••N interactions accounted for 11.1% of the crystal packing. The most common interactions were H•••H, which covered 38.4% of the mapped area, while π•••π interactions made up 3.3% of the crystal packing.
The surface morphology of 3a was investigated by combining Shape Index and Curvature fingerprint data(Fig. 5). SI is sensitive to any deviation from the lattice shape. The concave region on the upper plane for 6-Bromo-2-phenyl-1,3,4-triazaindan particle of the surface is marked by red triangles. The blue triangles indicate the position of the phenyl fragment on the outer superficial surface. The SI data matched the 2D pattern. The particle surface for 3a showed two patches of curvature due to the intermolecular connections.
3.4. Molecular modeling study
We used DFT/B3LYP/6-311G** to optimize the structures of regioisomers (3rd and 4th positions) and calculated their total energies with zero-point energy correction. The results are shown in Table 1. We observed that as expected [16], the 3rd position regioisomers were more stable than the 4th position ones when the alkyl group was small or distant. This was consistent with the experimental data.
Table 1
3.5. Electronic reactivity based on frontier molecular orbitals FMOs.
We used the DFT/B3LYP/6-311G** method to calculate the energy-gap “Δε” of 2a,2b,3a and 3b molecules based on their orbital distributions and the HOMO“electron-donor” and LUMO“electron-cceptor” levels (Fig. 6). The energy-gap “Δε” can indicate the kinetic stability of 2a,2b,3a and 3b molecules and its potential for chemical reactivity [37]. The 1,3,4-triazaindan system with a high “Δε” value has a high hardness “η” acts as a good nucleophile, while low “Δε” value for their system has a low hardness and acts as an excellent electrophile (Table 2). We also computed the ionization potential (IP), electronegativity “χ”, and global electrophilicity “ω” as other electronic properties related to the energy gap and presented them in Table 2.
The descriptor Δε was calculated for 1, 2a,2b,3a, and 3b for 6-Bromo-2-phenyl-1,3,4-triazaindan hybrids and compared with the reported values of biomaterials. The values of Δε ranged from 2.702 to 4.451 ev. for the compounds. The HOMO orbital was distributed over the phenyl-1,3,4-triazaindan fragment for 1, 2a,2b,3a, and 3b compounds, and it transferred intramolecularly to the LUMO orbital over the imidazole center (Fig. 6). The HOMO–LUMO orbitals showed electron cloud transfer within the phenyl-1,3,4-triazaindan skeleton in 2a,2b,3a and 3b, and had a shielding effect between the phenyl-1,3,4-triazaindan and the pyridine fragment. Moreover, 1, 2a,2b,3a, and 3b compounds had a low value of ω (between 7.5 and 15.8 ev.), indicating a high stabilization efficiency of the surface electrons. Compounds 2a and 3a had higher stabilization than 2b and 3b. The value of η indicated a low tendency for electronic current distortion. As expected[16, 38], the antimicrobial activity of the biomolecules was influenced by the antioxidant power, which was related to a low value of “IP” ionization potential[39]. The molecules had a scavenging ability due to the one-electron transfer mechanism and better antioxidants. The antioxidant power increased as the IP value decreased. The tested compounds had a low IP (3.7 to 6.04 ev.).
3.6. Fingerprint of the molecular electrostatic potential “MEP”
MEP stands for Molecular Electrostatic Potential, which is a measure of how the outer electrons of a molecule are distributed and how they affect the reactivity and interactions with H atoms of the molecular environment (Fig. 6).
Figure 6.
MEP also reveals the locations of electrophilic and nucleophilic sites on the molecule, which are important for chemical reactions. By using different colors to represent different levels of electrostatic potential, we can visually identify the polarity of the molecule. The red-area indicates a negative or polar charge, blue indicates a positive or nonpolar charge, and green-zone indicates a neutral or intermediate charge. The order of colors from red to yellow to blue to green reflects the change in electrostatic potential on the MEP diagram (Fig. 6). The distribution of electrons supports the idea that 1, 2a, 2b, 3a and 3b compounds can attack bacterial DHFR enzymes based on their size and shape. Figure 6 shows that the yellow region is concentrated over the 1,3,4-triazaindan ring in 1, 2a, 2b, 3a and 3b hybrids, which increases the electrophilicity effect. The blue section covers the 1,3,4-triazaindan substrate 1, 2a, 2b, 3a and 3b compounds, enhancing the nucleophilicity of the pyridine cores, which determines the ability of the substrate to recognize the binding site through electrostatic interaction with the receptor.
3.7. Molecular Docking Profile:
We used molecular docking analysis to investigate the biological activity of 1, 2a, 2b, 3a and 3b hybrids as potential antimicrobial agents. We docked these compounds into the active site of “DHFR”dihydrofolate reductase (PDB: 1DLS [40]) to examine their binding modes and conformations. We followed the docking steps described in previous studies [41, 42]. We generated the 3D loop of DHFR using mGen-THERADER and used it in the docking framework. This loop contains seven amino acid residues, which are important for maintaining the conformation of the enzyme. Moreover, it is believed that the DHFR active site is located within this loop, making it an ideal target for docking studies. We used binding-energy BE to study the toxicity of the 1,2a,2b,3a and 3b compounds with the DHFR receptor and compared them with the reference inhibitor (Methotrexate). We redocked the compounds and obtained a root mean square deviation (RMSD) less than 2 Å. The methotrexate interacted with important amino acids (GLU30, ILE7, VAL115, LYS 68, ARG 70, LYS68, ARG70, ILE7, and PHE34) in the DHFR binding site Fig. 7.
Figure 7:
The binding efficiency “ΔE” of 1,2a,2b,3a and 3b ligands was calculated based on the interaction fingerprint between the ligand and the DHFR (PDB: 1DLS) protein. The docking experiments were performed using the OPLS3e force field and the energy values are reported in Table 3. The best pose for each 1,2a,2b,3a and 3b ligands were selected based on the lowest energy “ΔE” and root mean square deviation (RMSD) values. The binding affinities of 1, 2a, 2b, 3a and 3b ligands were further evaluated by computing the “Ki” inhibition constant and the “LE”ligand efficiency [43]. Structurally of 1,2a,2b,3a and 3b ligands share a common 1,3,4-triazaindan scaffold, but differ in the alkylation site, which may affect their biological activity. “ΔE” values for 1,2a,2b,3a and 3b ligands with respect to dihydrofolate reductase (DHFR) varied depending on their structural features.
Table 3
The binding energy of methotrexate was revealed to be -7.85 kcal/mol with Ki = 1.88 A° via a sidechain of two H bonds with Arg91 and Ser92. The studied compounds' BEs were organized in increasing order as 1>2a> 3a >2a> 3a, with a promising constant inhibition range of 0.16 to 2.74. Compounds (2a and 3a) showed BE = − 7.44 and − 7.15 kcal/mol, respectively. These compounds were stabilized at the binding site via the arrangement of an imidazole ring with a perpendicular mode formed by a sticky π-π bond. 2-[2-(6-Bromo-2-phenyl-1,3,4-triaza-3H-inden-3-yl)ethyl]-2H-isoindole-1,3-dione (2a) with (Ala. l9 and Gly. 20) established two powerful H bonds. Furthermore, (3a) formed a separate important H bond with Ala9 (Fig. 7). The bioactivity indices LE and Ki were also all within the expected range [44, 45]. It is clear that molecular docking promoted antibacterial activity, which had an impact on the effectiveness of the docking experiment.
3.4. Biological activity
The pharmacological activities of several pharmaceutical preparations are generally linked to one of their region-isomers [46, 47], while most of these substances have several isomers, or at least one. After a hard work of synthesis to produce several derivates from the 1,3,4-Triaza-3H-indene; the last part of this work, aimed to valorize some of them by evaluating the antibacterial potential of N3-alkylated derivates of the 1,3,4-Triaza-3H-indene, 2a and 3a. For this purpose, two target strains were chosen to represent each bacterial type, namely E. coli as Gram-negative and B. cereus as Gram positive strain. The activities of the chosen molecules are listed in Table 3. The antimicrobial potential of the chosen synthesized molecules was evaluated thanks to the disk diffusion method, by measuring the inhibition zones diameters (Table 4).
Table 4
Both tested synthesized derivates were more active against Bacillus cereus than against Escherichia coli, which was far more resistant to the (3a) derivate and was sensitive only to the 2a derivate. The antimicrobial potential of different concentrations of the derivates was estimated against Bacillus cereus and Escherichia coli by determining the MIC values (Table 5), using the microdilution method.
Table 5.
As can be observed in Table 5, the two compounds possessed antimicrobial power to varying degrees depending on the microbial strain tested. Among the two tested strains, it is noted that B. cereus was most sensitive against all the studied compounds, which confirms the results obtained via the agar diffusion method. MICs values exhibited by antibiotic (Tetracycline) on the tested strain were 0.15 mg/mL and 0.7 mg/mL for B. cereus and E. coli respectively (Table 5). Similarly, the growth of B. cereus was inhibited by (2a) and (3a) products with a concentration exceeding 0.312 mg / mL (Table S.2, supplementary materials). By contrast, E. coli was the most resistant strain and was able to grow even at concentrations of 5mg/mL of synthesized product (3a). Only product (2a) was able to exhibit moderate inhibitory activity with MICs of 2.5 mg / mL.