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A new and suitable multicomponent one-pot reaction was developed for the synthesis of 2-amino-3-cyanopyridine derivatives. Background: This synthesis was demonstrated by the efficient and easy access to a variety of substituted 2-aminopyridines using enaminones as key precursors under solvent-free conditions. Methods: A range of spectroscopic techniques was used to determine and confirm the chemical structures (FTIR, 1 H NMR, 13 C NMR). The antimicrobial potency of synthesized compounds ( 2a &#; d ) was tested using disk diffusion assays, and the Minimum Inhibitory Concentration (MIC) for the active compounds was determined against a panel of microorganisms, including Gram-positive and Gram-negative bacteria and yeasts. Moreover, a docking analysis was conducted by Molecular Operating Environment (MOE) software to provide supplementary information about the potential, as well as an ADME-T prediction to describe the pharmacokinetic properties of the best compound and its toxicity. Results: The results of the antimicrobial activity indicated that compound 2c showed the highest activity against Gram-positive bacteria, particularly S. aureus and B. subtilis whose MIC values were 0.039 ± 0.000 µg·mL &#;1 . The results of the theoretical study of compound 2c were in line with the experimental data and exhibited excellent antibacterial potential. Conclusions: On the basis of the obtained results, compound 2c can be used as an antibacterial agent model with high antibacterial potency.
Recently, many methods for the synthesis of 2-aminopyridines have been developed. However, the MCRs used for the preparation of substituted 2-aminopyridines are very limited. In the continuation of previous work regarding the field of aminopyridine derivates, the present study aims to synthesize new MCRs using enaminones with a simple process of exploring their antimicrobial profile in vitro and computational studies.
As a part of our interest in the synthesis, we reported a number of clean approaches to prepare building blocks made of 2-aminopyridines [ 8 ], 2-pyridones [ 9 ], chromenopyridines [ 10 ], and pyrimidopyridines [ 11 ] from different enamines. Based on these data, multicomponent reactions (MCRs) in a one-pot process give a single product which represents a powerful tool in modern medicinal chemistry. This unique strategy leads to the formation of many new bioactive compounds due to their convergence, rapid process, minimum waste production, and high yields [ 12 ].
Several pyridine compounds are well known and present in many medicinal preparations with various biological profiles, to name only the 2-aminopyridine derivatives ( ) considered precursors for the synthesis of a variety of heterocyclic compounds [ 1 ]. Among them, 2-aminopyridines derivatives ( ) have been considered precursors of the synthesis of a variety of heterocyclic compounds [ 1 ] and represent effective agents for their antibacterial [ 2 ] anticancer [ 3 ], anti-inflammatory [ 4 ], Ketohexokinase (KHK) inhibitory [ 5 ], and NO synthases inhibitory activities [ 6 ] ( ). Nowadays, microbial resistance represents one of the most world&#;s intricate problems due to the increasing rate of mortality and morbidity [ 7 ], hence making the synthesis of new compounds to fight multidrug microorganisms with broad activity extremely challenging.
This section is divided into subheadings to describe briefly and precisely the experimental results and their interpretation, as well as draw experimental conclusions.
Our research group developed new synthetic methodologies for the synthesis of both building blocks and heterocyclic compounds under free solvent conditions. In the previous paper, we reported an efficient method to obtain enaminones as key building blocks from the condensation of methyl ketones with dimethylformamide dimethyl acetal (DMFDMA). Thus, in continuation of our interest in the development of new methodologies, in this article, we describe the synthesis of 3-cyano-aminopyridines in two steps. The synthesis of the target compounds was carried out as illustrated in .
Open in a separate windowThe key substrate enaminones 1a&#;c are also very versatile intermediates for the synthesis of many heterocycles.The products 1a&#;c were prepared according to our previous works from acetophenone and an equimolar amount of dimethyl acetal dimethyl-formamide (DMFDMA) under microwave (MW) irradiation and without solvent to obtain 1a&#;c with excellent yields (80&#;86%) ( ).
The experimental conditions used for the synthesis of 2-amino-3-cyanopyridines were optimized by using enaminone 1, malononitrile, and benzylamine ( , entry 1). Initially, the reactants were introduced without any solvent, and the reaction was allowed to proceed at room temperature for 24 h. However, no products were isolated under these conditions (entry 1). At 40 °C and for 24 h, the reaction provided product 2b with a yieldof 20% (entry 2). When the temperature was evaluated at 60 °C, the yield of 2b was increased to 40% (entry 3) for 6 h. The best yield was obtained at 80 °C for 3 h. The structure of 2b was identified by spectroscopic analysis.
From the optimized reaction, the generality of this MCR was explored using different enaminones 1a&#;c and primary amines ( ) to provide the corresponding products 2a&#;l. It was found that the synthesis under solvent-free 2-aminopyridine derivatives can be obtained by a simple, fast, and cleaner method using the three-component reaction. Given the exceptional biological properties of heterocyclic aminopyridines, a wide variety of primary amine and enaminones were evaluated, which provided a convenient and flexible method for the synthesis of 2-aminopyridines.
The proposed mechanism showed that the enaminone reacts at first with malononitrile via Knoevenagel reaction to afford intermediate I. However, the intermediate I reacted with primary amines at the nitrile groups of intermediate II then the product inter-cyclized to give intermediate III. The reaction is finished with an aromatization step to afford the 2-aminopyridine structure VI ( ).
Open in a separate windowThe structureof 1a was confirmed by NH characteristic absorption bands in the IR spectrum of the starting enaminones 1a&#;c, and the 1H NMR spectra of the same derivatives showed two singlet signals corresponding to NCH3 protons at δ 2.94 and 3.01 ppm. The formation of compounds 2a&#;l was confirmed by spectral data and elemental analyses. The 1H NMR spectra of these derivatives demonstrated the appearance of a new doublet signal at 6.69 ppm corresponding to the CH=CH protons. Mass spectra of these compounds showed distinctive molecular ion peaks at the right m/z values. The IR spectra of these compounds showed the characteristic NH stretching bands in the range of &#; cm&#;1. In addition, the 1H NMR spectra of the same derivatives showed singlet signals corresponding to NH protons at δ 5.11&#;5.11 ppm.
The antimicrobial activity of each chemical compound was initially tested against twelve target microorganisms. A disk diffusion data assay was employed for primary screening. As shown in , the highest antimicrobial effect was observed against Gram-positive bacteria, particularly against rod bacteria, including Bacillus subtilis ATCC, Listeria monocytogenes ATCC , and Bacillus cereus ATCC . The inhibition zone diameters ranged between 11.33 ± 0.57 mm and 13 ± 0 mm. Compared with the Gentamicin inhibition zone diameter, Listeria monocytogenes ATCC was found topossess a similar diameter using a Gentamicin half-concentration. Moderate activity against target cocci Gram-positive bacteria was observed, and the diameters of the inhibition zones were between 8.66 ± 0.57 mm to 9.66 ± 0. However, no effect was observed against Gram-negative bacteria and two yeasts. These results were obtained from compound 2c, while no activity was detected from 2a, 2b, and 2d, which represented the compound 2c analogs. These results can suggest the following: (i) antimicrobial activity is due to cyclohexylamine presence, and (ii) in a series of 2-aminopyridine compounds, the introduction of other CH2 after amine function was responsible for the activity disappearance.
Compound 2c&#;s selective activity against only Gram-positive bacteria can occur due to the cell&#;s structure. It is known that Gram-positive bacteria are more sensitive than Gram-negative ones [13] because the cell structure of their wall, with a principal share of peptidoglycan, p enables the hydrophobic compounds to infiltrate the cells and proceed on the wall as well as on the cell membrane and inside the cytoplasm. The cell structure of the Gram-negative bacteria&#;swall is more complex with a reduced amount of peptidoglycan and with an external membrane composed of a phospholipids&#; double-layer connected with the internal membrane by lip polysaccharides [14]. Therefore, Gram-negative bacteria present a further complex biological barrier that avoids the penetration of different antibiotics, whereas their periplasmic space has proteins and enzymes that are able to break down foreign molecules [15].
The disk diffusion assay is the primary screening representing a rapid and qualitative method to detect the antimicrobial potency of compounds. For a quantitative assessment, the MIC assay was done using the active chemical compound 2c, and the results are listed in . Overall, the most Gram-positive bacteria-sensitive strains were S. aureus and B. subtilis, with MIC values of 39 ± 0.000 µg·mL&#;1. B.cereus, E. faecalis, and M. luteus expressed sensitivity with MIC values of 78 ± 0.000 µg·mL&#;1. However, L. monocytogenes exhibited a moderate sensitivity with a MIC value of156 ± 0.000 µg·mL&#;1 in comparison to the MIC values of the standard drug Gentamicin.
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These data are in agreement with Chikhalia&#;s and Patel&#;s results [16], which indicated the presence of cyclohexylamine generated antibacterial activity towards Gram-positive bacteria. However, MICs values were significantly higher compared to those of the present study.
The mechanism responsible for the selective antibacterial profile of the 2c compound is unknown at the moment, and then we speculated that the antibacterial potency might have been related to the global charge of the compound, in particular to the positive charge of carbon directly connected to the cyclic compound. The cationic head groups bind and disrupt the bacterial cell membrane with the aid of hydrophobic and electrostatic interactions leading to the release of cytoplasmic constituents and the death of the cell [17]. Work is in progress to provide a detailed clarification as to theantibacterial action mechanism of the effective compound.
The detailed results of compound 2c&#;s docking simulation with both X-ray crystals of S. aureus (PDB ID: 4URM) and B. subtilis (PDB ID: 2RHL) targets aresummarized in . To estimate all possible interactions, the docking outputs generated by MOE software were converted into (.pdb) files and visualized with the default parameters of the BIOVIA DS visualizer package (Dassault Systèmes BIOVIA, Discovery Studio Modeling Environment, ).
In general, compound 2c&#;sdocking results demonstrated a good interaction with two target proteins, the S. aureus (PDB ID: 4URM) and B. subtilis (PDB ID: 2RHL) targets. This confirms that compound 2c fits well in the binding pockets of these bacterial targets.
According to the docking results ( ), compound 2c is identified to bind to the S. aureus ATP binding pocket with a score value very close to native KBD (kibdelomycin) (&#;5.532 vs. &#;6.383 kcal/mol)). In addition, it was noted that this compound established the interactions with B. subtilis pocket with a score value very close to native GDP (kibdelomycin) (&#;6.389 vs. &#;7.843 kcal/mol)).
As presented in , compound 2c binds to the same site as KBD and GDP, but with different residues, respectively. It is stabilized by several hydrogen-bonding interactions and close hydrophobic contacts with surrounding amino acids.
Open in a separate windowBased on the antimicrobial assay results ( and ), compound 2c represents the activity against S. aureus and B. subtilis and established several interactions with the active site residues of S. Aureus (PDB ID: 4URM) and B. subtilis (PDB ID: 2RHL) targets.
The compound 2c&#;s docking conformation showed that itestablished five interactions with S. aureus active site residues (PDB ID: 4URM). Two strong conventional H-bonds [18,19] were observed: the first one was observed between the hydrogen atom of the compound and active site residues GLU(A:58) with a bond distance of 2.62 Å, and the secondwas established between the oxygen atom of the compound and the active site residues THR(A:173) with a bond distance of 2.88 Å. We can clearly see these two residues: GLU(A:58) and THR(A:173), play an important role in the active site of S. aureus (PDB ID: 4URM), which have been reported inprevious studies [20,21].
A Pi&#;Sigma interaction formed with active site residues; ASN(A:54) has a bond distanceequal to 2.38 Å ( ). Two other weak interactions of hydrophobic types appeared: the first is of the type Pi&#;alkyl and was established with six rings of the compound and active site residues ILE(A:86) with a bond distance of 4.76 Å, and the second, which is of the type Alkyl formed the carbon atom of the compound and active site residues PRO(A:87) with abond distance of 4.62 Å ( and ). Furthermore, Rahman, M. et al. [22] and Pham, E.C. et al. [23] confirmed that ASN(A:54), PRO(A:87) and ILE(A:86) are responsible forthe formation of different interactions in the binding site of the target.
In addition, compound 2c&#;s docking results showed good interactions with the second target (B. subtilis), especially because it established five interactions with B. subtilis active site residues (PDB ID: 2RHL).Three strong conventional H-bonds [18,19] were observed between the nitrogen atom of the compound and three active site residues GLY(A:110), GLY(A:108), and THR(A:109) with bond distances of 2.38 Å, 3.10 Å, and 3.10 Å, respectively. We note that these results were confirmed by Singh, D. et al. [24]. Pi&#;cation interactions formed between six rings of the compound with active site residues ARG(A:143) with a bond distance of 3.96 Å ( ). Pi&#;alkyl type is another weak interaction formed between the carbon atom of the compound and active site residues PHE(A:183) with a bond distance of 5.18 Å ( and ). In addition, Matsui, T. et al. [25] confirmed that ARG(A:143) and PHE(A:183) are the keysto the majority of interactions. Finally, fora comparison, compound 2c&#;s binding mode in the pocket sites of the S. aureus (PDB ID: 4URM) and B. subtilis (PDB ID: 2RHL) targets, and in line with our expectations, compound 2c is predicted to bind to the active site residues of both bacterial targets with high affinities compared to the native compounds.
According to the docking studies, there was sufficient evidence that compound 2c forms several H-bond interactions with S. aureus (PDB ID: 4URM) and B. subtilis (PDB ID: 2RHL) targets and should exhibit the inhibition of both bacterial targets. However, this compound showed strong inhibitory activities when tested against Gram-positive bacteria, particularly against S. aureus and B. subtilis, ofwhich the MIC values were 0.039 ± 0.000 µg·mL&#;1. This might be one of the reasons this compound showed biological activity and different interactions with both target proteins.
Drug-likeness and physicochemical properties of compound 2c were evaluated by checking Lipinski&#;s rule of five and the Veber and Ghose rules, which are crucial for rational drug design. In addition, absorption, distribution, metabolism, excretion, and toxicity (ADMET) are crucial parameters for the drug development process. All results are given in .
It is apparent from this table that compound 2c has a number of hydrogen bond donors < 7 (n-HD: (0~7)) and hydrogen bond acceptors < 12 (n-HA: (0~12)).Furthermore, the molecular weight of the compound belongs to the interval: 100~500 g/mol, and the MLogPand WLogP values are <5. Further analysis of the table revealed that the compound 2c exhibited three rotational bonds (nROTB) (should be <10) and a topological polar area value (TPSA) of 48.71 Å (less than 140 Å), justifying the flexibility of the molecule and its good permeability in the cellular plasma membrane to cross the blood&#;brain barrier (BBB).
In addition, this result indicates that compound 2c satisfies all of the criteria of drug-likeness without any violation of the Lipinski, Veber, and Egan rules.
According to the literature [26,27], the process of drug development must use suitable parameters that represent the ADMET properties. An effective candidate drug may sometimes be effective against the therapeutic target (affinity and selectivity), but this is not sufficient&#;it must be safe and have adequate ADME qualities in a therapeutic concentration.
As can be seen from the above table, compound 2c has an average Caco-2 permeability, and HIA value is higher than 30%, suggesting that the compound can be administered orally and is strongly absorbed by the gastrointestinal system into the bloodstream. We also note that the logPS value of the compound is between &#;2 < logPS < 0, which means that compound 2c is able to penetrate the CNS. Additionally, the logBB value of this compound is 0.22; this indicates that the compound presents an average distribution in the brain ( ).
The inhibition of one of the cytochrome P450 (CYP) isoforms may alter drug metabolism, while a lack of inhibition may mean that the compounds will not alter the metabolism of other substances [28]. Compound 2c will interact with some Cytochrome P450 isoforms (CYP1A2 and CYP2D6) with the exception of CYP2C19. In addition, this compound is not likely to obstruct the organic cation transporter substrate (OCT2), and it has a low excretion time. Finally, the compound is not likely to be cardiotoxic as it does not obstruct the hERG K+ channels linked to deadly cardiac arrhythmias [29], and no AMES toxicity was found ( ).
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