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New N-amino-5-cyano-6-pyridones as antimicrobial small molecules endowed with DNA gyrase a inhibitory activity: design, one-pot synthesis, biological assessment and in silico insights

BMC Chemistry volume 18, Article number: 226 (2024) Cite this article

Abstract

A set of innovative N-amino-5-cyano-6-pyridones derivatives was developed and produced using one-pot three-component procedures. The evaluated molecules were examined for their antimicrobial efficacy. Based on the acquired findings, most of the investigated compounds had promising antimicrobial properties. Out of these derivatives of 3-cyanopyridine, compounds 3d and 3e exhibited minimum inhibitory concentrations (MIC) of 3.91 µg/mL against E.coli. In vitro evaluation of DNA gyrase A displayed that molecule 3d exhibited promising potency as an inhibitor, with an IC50 value of 1.68 µg/mL compared to ciprofloxacin (IC50 = 0.45 µg/mL). Furthermore, it was observed that molecule 3e exhibited a moderate inhibitory effect, as indicated by its IC50 value of 3.77 µg/mL. A kinetics study conducted to assess the time required to kill E. coli bacteria demonstrated that gentamycin and compounds 3d and 3e exhibited bactericidal effects within a time frame of 90–120 min. Based on the ADME predictions, compounds 3d and 3e are expected to have favorable oral bioavailability and are unlikely to penetrate the blood-brain barrier. Computational mutagenicity and tumorigenicity studies were conducted on compounds 3d and 3e. The molecular docking investigation has conclusively demonstrated the binding of compounds 3d and 3e to the target DNA gyrase A enzyme, further reinforcing the existing data.

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Introduction

In developing countries, there is a high incidence of microbial infections, which are leading to a rising number of illnesses and deaths. The global rise of antibacterial resistance poses an increasingly significant danger to human health [1]. The pace of finding new antibacterials is significantly surpassed by the speed at which resistance is growing, and there is a pressing demand for the creation of novel medicines for antibacterial purposes [2, 3]. Developing countries are particularly worried about the over-abuse of antibiotics in human as well as animal communities. Consequently, these circumstances result in the physician having fewer treatment options, longer hospital stays, higher costs, greater vulnerability to infection, and elevated mortality rates [4]. Antibiotic resistance poses a significant risk to the successful prevention and treatment of a growing range of bacterial diseases, rendering it one of the most prominent obstacles to worldwide medical systems. Presently, an estimated 700,000 individuals globally succumb to drug-resistant diseases annually [5]. N-substituted 2-pyridones are nitrogen-containing heteroarenes that have antimicrobial properties, rendering them appropriate for several medical uses [6]. The FDA database contains over 50 drugs derived from pyridine, such as pyridostigmine for myasthenia gravis, omeprazole for peptic ulcer, lornoxicam for arthritis, amlodipine for hypertension, milrinone for cardiac support, torsemide as a diuretic, bromazepam for anxiety, tacrine for Alzheimer’s disease, abemaciclib for breast cancer, and omidenepag for glaucoma [7, 8]. Cyanopyridine derivatives are crucial intermediates in the synthesis of biologically active compounds, thus they play an important role in chemical synthesis [9]. Pharmaceuticals greatly benefit from the utilization of compounds that incorporate the pyridine-3-carbonitrile group [10]. Figure 1 displays the effectiveness of pyridine/Pyridone as an antimicrobial agent. Specifically, 6-dimethyl-2-oxo-pyridine-3-carbonitrile I demonstrated an antibacterial effect against Escherichia coli (IZ = 22.8 mm) and Pseudomonas aeruginosa (IZ = 21.4. mm) close to that of the reference streptomycin (IZ = 25 mm against E. coli and 24 mm against P. aeruginosa) [11]. Furthermore, it was discovered that the compound II, a derivative of 1,4-dihydropyridine, exhibited significantly higher activity (IZ = 25 mm) in comparison to the conventional ciprofloxacin (IZ = 22 mm) against S. aureus [12]. Moreover, the presence of the pyridine ring in molecule III played a crucial role in its effectiveness towards C. albicans. The minimum inhibitory concentration (MIC) result for this molecule III was 15.6 mg/mL [13]. Moreover, pyridine-3-carbonitrile compound IV demonstrated a comparable inhibitory activity towards E. coli (MIC = 0.013 µM) to the reference medication amoxicillin (MIC = 0.01 µM) [14]. Moreover, molecule V had remarkable efficacy towards gram-negative bacteria, specifically E. Coli, with an IZ measuring 22 mm, while the reference ofloxacin showed IZ value of 24 mm [15]. The pyridone derivative VI showed significant antibacterial activity toward B. subtilis, and S. aureus (MIC = 0.078 and 0.0024 mg/mL, respectively) compared to cefaclor (MIC = 0.0024 mg/mL) [16]. Further, Compound VII exhibited a wide range of antibacterial activity (MIC of 1.95 µg/mL for Streptococcus pneumonia, 0.98 µg/mL for B. subtilis, and 1.9 µg/mL for Salmonella typhimurium) that was equipotent to the reference drugs ampicillin (against Streptococcus pneumonia and B. subtilis) and gentamycin (against Salmonella typhimurium). Furthermore, it demonstrated antifungal activity towards Aspergillus fumigatus and C. albicans (MIC values of 1.95 µg/mL and 7.81 µg/mL, respectively) relative to the antifungal reference amphotericin B (MIC = 1.95 µg/mL) [17]. Compound VIII demonstrated potent antibacterial effects against methicillin-resistant S. aureus equal to that of vancomycin (MIC = 1 µg/mL) [18]. In addition, compound IX exhibited strong antifungal activity equivalent to that of nystatin against C. albicans (MIC = 1.95 µg/mL) [19]. Furthermore, 2-pyridones are important class of antimicrobial compounds because of their efficacy against the bacterial type II DNA topoisomerases (DNA gyrase and topoisomerase IV). Also, 2-oxopyridine derivatives are isomeric to 4-oxopyridine core, which is present in the fluoroquinolone class of antibiotics [20]. From the reported DNA gyrase inhibitors, compounds X and XI displayed good anti-gyrase activity (IC50 = 21.97 and 39.15 µM, respectively) compared to the reference ciprofloxacin (IC50 = 26.32 µM) [20]. As well, the 3-cyanopyridine derivative XII (IC50 = 0.44 µM) showed nearly equal activity against DNA gyrase to ciprofloxacin (IC50 = 0.42 µM), while compound XIII exhibited half the activity of ciprofloxacin (IC50 = 0.94 µM) [21]. Furthermore, compound XIV containing fused pyridine core demonstrated DNA gyrase inhibitory activity (IC50 value of 7.35 µΜ), which was more potent than Ciprofloxacin (IC50 value of 47.68 µM) [22].

Fig. 1
figure 1

Reported pyridine/Pyridone as antimicrobial compounds

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Our research focused on synthesizing novel nicotinonitriles to explore their antibacterial properties. We assessed their antimicrobial activity by determining their IZ (inhibition zone) and MICs (minimum inhibitory concentrations). The potential compounds that have demonstrated strong antibacterial effects will undergo additional investigation using in vitro enzyme potency assessment for DNA gyrases A, in order to examine their antibacterial properties. Additionally, a bacterial killing kinetic assay was performed to evaluate the antibacterial activity of the most active compounds over time and to predict their safety profile through assessing their cytotoxic effect on normal cells using the MTT technique. The study will involve conducting docking analyses to investigate the binding affinities of the enzyme of interest, as well as performing in silico ADME forecasting to assess the physicochemical properties.

Rationale and design

The optimization approaches for the compound I depend on the strategies shown in Fig. 2. Compound I exhibited significant antibacterial properties towards Pseudomonas aeruginosa and E. coli with IZ 21.4, and 22 mm, respectively [11]. The objective of this work was to substitute the methyl group at position 4 with aryl moiety, explore the impact of different substituents, and replace the methyl group at position 6 of compound I with a hydrophilic amino group at position 2, which could potentially participate in hydrogen bonding. Furthermore, our objective was to preserve the N-aminocynopyridone ring. Carboxamide or carbothiomide, which may also be significant in the formation of hydrogen bonds, is additionally introduced.

As the N-aminocynopyridone ring is considered the pharmacophoric moiety responsible for the stated activity, our research focused on synthesizing a variety of new derivatives. Specifically, we aimed to develop derivatives based on 6-oxo-1,6-dihydropyridine-3-carboxamide/carbothioamide. We will evaluate the antimicrobial properties of all compounds against two strains of fungi, two strains of Gram-negative bacteria, and two strains of Gram-positive bacteria. The most active molecules have been investigated for inhibition assays of DNA gyrase A, and time-killing kinetics research against E. coli was conducted. To determine the biological activity of these molecules, we select the most potent ones and analyze their ADME properties using the SwissADME, Molsoft, PreADME, and Datawarrior websites. Additionally, docking studies will be performed on DNA gyrase A.

Fig. 2
figure 2

The design of new cyanopyridones

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Results and discussion

Chemistry

This investigation describes a one-step reaction in which diverse aldehydes react with cyanoacetohydrazide, cyanoacetamide, or cyanothioacetamide. Using this approach produced N-amino-5-cyano-6-pyridones in high yields through a multicomponent reaction, as Scheme 1 shows. Firstly, equimolar ethyl cyanoacetate was added to hydrazine hydrate at 0 °C while stirring to produce cyanoacetohydrazide 1. A 95% yield was obtained by filtering, rinsing with ethanol, and drying the solid cyanoacetic acid hydrazide 1 [23]. The synthesis of derivatives of 1,2-diamino-5-cyano-6-oxo-1,6-dihydropyridine-3-carboxamide 3a-e was carried out by heating cyanoacetohydrazide 1, cyanoacetamide 2a, and different aldehydes in a mixture of ethanol and water, with the addition of a small quantity of piperidine as a catalyst [24]. Subsequently, the compound cyanothioacetamide 2b was combined with cyanoacetohydrazide 1 and various aldehydes. Following this, the mixture was heated at 80 °C under reflux in a solution of ethanol and water. Additionally, a small quantity of piperidine was added as a catalyst. The aforementioned reaction led to the synthesis of the needed compounds 4a-d [24]. The molecular structures of the newly produced molecules 3a-e and 4a-d were determined based on their infrared (IR), proton nuclear magnetic resonance (1H NMR), carbon-13 nuclear magnetic resonance (13C NMR) spectroscopic, mass spectrometric data, and elemental analyses. These data were found to be in complete agreement with the hypothesized structures. The IR spectra of derivatives 3a-e showed typical bands corresponding to the NH2 groups in the region of (3396 − 3185) cm− 1. As an illustrative example, the spectrum of 3a was examined. The infrared spectrum exhibited bands at 3339, 3315, and 3236 cm− 1 (indicating the presence of 3NH2), 2207 cm− 1 (indicating CN), and 1680, 1669 cm− 1 (indicating 2 C = O). The 1H NMR spectrum revealed a new singlet at δ 2.93 ppm, which corresponds to CH3 protons. Additionally, there were D2O-exchangeable signals at δ 5.86, 7.34, and 7.69 ppm, suggesting the existence of three NH2 protons. The structure was determined by analyzing the MS spectrum, which showed a molecular ion peak [M+, 25.52%] at m/z 207. The infrared spectrum of 4c represented distinct bands at specific wavenumbers, namely 3392, 3352, and 3199 cm− 1 (corresponding to the existence of 3NH2), 2223 cm− 1 (indicating the presence of CN), and 1673 cm− 1 (representing C = O). The 1H NMR spectrum of compound 4c showed a singlet signal at δ 3.04 ppm, which was attributed to two CH3 groups. Additionally, there were three signals at δ 7.63, 8.49 and 9.67 ppm that could be exchanged with D2O, corresponding to three NH2 protons. The 13C NMR spectra of 4c showed a signal at δ 189.93 ppm, which corresponds to the C = S carbon. The MS spectrum displayed the structure of the compound, featuring the ion peak of the molecule [M+, 5.01%] at m/z 328.

Scheme 1
scheme 1

Synthesis of molecules 3a-e and 4a-d; Reagents and conditions: (i) Ethanol/ H2O, piperidine, stirring at 80 °C, 4 h; (ii) acetaldehyde, (iii) 2,4- dichlorobenzaldehyde, (iv) N-dimethylaminobenzaldehyde, (v) 4-(benzyloxy)benzaldehyde, (vi) 4-formylphenyl acetate

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Biological studies

The novel molecules were evaluated for antimicrobial properties utilizing the agar well diffusion method against six different microorganisms. The selected pathogenic microorganisms included Bacillus pumilus and Streptococcus faecalis as Gram-positive bacteria, Enterobacter cloacae and E. coli as Gram-negative bacteria, and Saccharomyces cerevisiae and Candida albicans as fungi. Penicillin G served as the reference antibiotic for Gram-positive bacteria, whereas ciprofloxacin was the reference antibiotic for Gram-negative bacteria. Ketoconazole was utilized as the reference antifungal medicine. The broth dilution method was utilized to ascertain the minimum inhibitory concentration (MIC), and minimum bactericidal/ fungicidal concentration (MBC/MFC).

Antimicrobial testing

The obtained preliminary antimicrobial test results (Table 1& Fig. 3) demonstrated that the investigated compounds demonstrated favorable outcomes against specific bacteria and fungus strains. Compound 4a, which contained 3-carbothioamide and 4-methyl groups attached to the cyanopyridine nucleus, exhibited significant broad-spectrum antimicrobial activity against the tested bacteria and fungi. Inhibition zones (IZs) for compound 4a ranged from 16 to 23 mm. In contrast, compound 3a, which contained 3-carboxamide groups, exhibited diminished activity against all tested bacteria and fungi, with the exception of E. cloacae (IZ = 21 mm). With inhibition zones (IZs) ranging from 18 to 22 mm, compounds 3b and 3d exhibited a notable antibacterial impact against B. pumilus, S. faecalis, and E. coli. In contrast, these compounds demonstrated only mild to moderate activity against E. cloacae and two fungal strains, with IZs ranging from 9 to 16 mm. Additionally, 4b bearing di-chlorophenyl at position 4 of pyridine-carboxamide, was highly active only towards E. coli, S. faecalis, and E. cloacae (IZs = 18 &19 mm) and weak against tested fungi. Furthermore, compounds 3c and 4c displayed high antimicrobial activity on E. coli with inhibition of 20 and 23 mm, respectively and these compounds had mild antimicrobial activity towards other bacterial and fungal strains. Finally, compounds 3e and 4d exhibited mild to moderate antimicrobial activity towards tested bacteria and fungi strains.

Table 1 Inhibition zones (mm) of tested molecules towards fungal and bacterial strains
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Fig. 3
figure 3

Inhibition diameter zones in millimeter

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Evaluation of MIC, MBC, and MFC

The results for the minimum inhibitory concentrations (MIC), the minimum bactericidal concentrations (MBC), and minimum fungicidal concentrations (MFC) were determined for further investigation. The MBC/MIC and MFC/MIC ratios were determined. The obtained results are illustrated in Tables 2 and 3.

MIC and MBC

Regarding Gram-positive Strains: All the analogs exhibited significantly higher inhibitory potency against certain gram-positive bacteria, in comparison to penicillin G as a reference drug. Besides, derivative 4d bearing phenoxyphenyl at position 4 of pyridine-carbothioamide groups was the most potent antimicrobial against B. pumilus, they elicited 8 times more potency than the standard Penicillin G (MIC; 7.81 vs. 62.5 µg /mL). Furthermore, the activity of compounds 3b-d was found to be four times that of the reference, penicillin G (MIC: 15.6 vs. 62.5 µg/mL). Compounds 3a and 4c were 4 times more potent antibacterial against S. faecalis than the reference, penicillin G (MIC; 7.81 vs. 31.5 µg /mL). Carboxamide derivatives exhibited a higher degree of antimicrobial efficacy against the tested strains, particularly the tested gram-negative bacteria, compared to carbothioamide derivatives, as seen in Fig. 4.

Fig. 4
figure 4

SAR of the antimicrobial activity of 3a-e and 4a-d

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The study aimed to demonstrate the antimicrobial activities of the examined derivatives, which were found to be bactericidal. The bactericidal effect was observed when the minimum bactericidal concentration was no more than four times the minimum inhibitory concentration value [25, 26]. Our derivatives exhibited a significant MBC/MIC ratio of roughly ≤ 4, showing their strong bactericidal activity. Compounds 3b, 3e, and 4a-c exhibited highly effective bactericidal activity against B. pumilus, with MBC values ranging from 31.3 to 125 µg/mL. The MBC/MIC ratios for these compounds were 2, indicating a strong bactericidal effect. In comparison, penicillin had an MBC of 125 µg/mL and an MBC/MIC ratio of 2. Compounds 3a, 3c, 3d, and 4d, on the other hand, had MBC/MIC ratios of 4. Furthermore, derivatives 3b, 3c, and 3e, along with derivative 4d, demonstrated enhanced bactericidal efficacy against S. faecalis, as evidenced by their respective MBC values of 62.5 and 125 µg/mL. The ratio of MBC to MIC was 2. It is worth mentioning that the newly synthesized compounds showed strong bactericidal activity towards E. coli and E. cloacae with MBC/MIC ratios: 2 and 4 except compound 3a which has an MBC/MIC ratio equal to 8.

Structure-activity relationship study

Table 2 present a summary of the MIC values of Carboxamide and carbothioamide derivatives (3a-e and 4a-e) against selected gram-negative and gram-positive bacteria in addition to two types of fungi. Most of our compounds exhibited a promising activity against E.coil. Based on the acquired MIC values against E.coil, it can be concluded that carboxamido compounds 3d and 3e exhibited the highest efficacy of E. coli (MIC = 3.91 µg/mL). In contrast, compound 4d demonstrates 7.81 µg/mL against the E.coli. Substituting benzyloxy group in compound 3d with an electron-donating dimethyl amino group in compound 3c results in a decrease in activity against E.coli, as seen by an increase in MIC value from 3.91 µg/ml to 15.6 µg/ml. Carboxamide compound 3c, which possesses stronger electronegative atoms (dichloro) exhibited MIC value (15.6 µg/mL) two-folds that of its counterpart carbothioamido 4c (31.3 µg/mL). To summarize the relation between the effect of the substitution at position 4 within carboxamides 3a-e, the anti-E-coli activity was increased in the order of benzyloxy phenyl = phenyl acetate > 2,4di-Cl benzene > CH3 = N-dimethylaminonenzene. Finally, the effect of 4 substitution on the anti-E-coli activity of carbothioamido derivatives 4a-d increased in the following order: 2,4-di-Cl benzene = benzyloxy phenyl > CH3 > N-dimethylaminonenzene.

Table 2 In vitro MIC, MBC (µg/mL), and MBC/MIC ratio of the tested molecules
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MIC and MFC

The compounds that were evaluated showed generally week efficacy against the fungus strains that were studied (Table 3). Substantial fungicidal effectiveness against C. albicans was demonstrated by compound 4c, which had a MIC of 15.6 µg/mL and MFC to MIC ratio of 4. Concurrently, compounds 3a, 3c, 3d, and 4d demonstrated noteworthy fungicidal efficacy towards C. albicans, as evidenced by their MIC values of 31.3 µg/mL. The MIC to minimal fungicidal concentration (MFC) ratio was 2, while the MFC was 4.

Table 3 In vitro MIC, MFC (µg/mL), and MFC/MIC ratio of the tested molecules
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Inhibitory effects against gyrase A enzyme

The DNA gyrase A undergoes negative supercoiling due to the activity of an enzyme. It was proposed that it has a role in transcription and replication in both the initiation and elongation stages in bacterial species [27]. As a result, the inhibition of its activity by a new inhibitor presents a special advantage concerning the emergence of bacterial resistance. The inhibitory behaviors and mechanisms of the most active derivatives, 3d and 3e, against DNA gyrase A isolated from E. coli were assessed. Table 4 displays the acquired data as IC50 values of enzyme inhibition in µg/mL. The MICs and IC50 exhibited a strong association (Tables 2 and 4), suggesting that these derivatives’ inhibition of DNA gyrase resulted in the suppression of bacterial cell growth. The derivatives 3d and 3e, which were the most active, demonstrated the ability to inhibit DNA gyrase, with IC50 values of 1.68 and 3.77 µg/mL, respectively.

Table 4 In Vitro inhibitory activities of the tested molecules 3d and 3e against DNA gyrase a enzyme
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The in vitro cytotoxicity of compounds 3d and 3e on the normal WI38 cells was examined as well using the MTT assay to evaluate their safety profiles. Table 4 shows that both substances, 3d (IC50 48.8 µM) and 3e (IC50 43.2 µM), were safer than the standard drug, ciprofloxacin (IC50 23.9 µM).

Time-kill kinetic test

The kinetics of derivatives 3d and 3e’s killing of bacteria against E. coli were examined in order to investigate their antibacterial activity in more detail (Fig. 5). Compound 3d exhibited bactericidal activity against E. coli after a duration of 90 min. Furthermore, during 120 min of incubation, molecules 3e and gentamycin (1x MIC) demonstrated notable bactericidal activity against E. coli.

Fig. 5
figure 5

Time-kill kinetics of molecules 3d, 3e, and gentamycin at 1xMIC towards E. coli

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Molecular docking and in silico calculations

In silico prediction of physicochemical, pharmacokinetic, mutagenicity, tumorigenicity studies, and solubility value

Pharmacokinetic data obtained in silico offer preliminary but important insights into how a drug will ultimately be metabolized by the human body. An oral pharmaceutical candidate is considered to meet Lipinski’s criterion if it has a LogP value of 5 or less, a molecular weight (MW) of 500 or less, no more than 5 hydrogen bond donors, and no more than 10 hydrogen bond acceptors [28]. Furthermore, polar surface area (TPSA) rather than the quantity of hydrogen bonding groups could be taken into consideration. Rats with TPSA of less than 140 Å2 ought to have good oral bioavailability. The pharmacokinetics of the most active drugs, 3d and 3e, were assessed using SwissADME software: [29], Molsoft [30], Pre-ADMET [31] and Data Warrior [30]. It was discovered that there is a Lipinski zero violation in the two compounds’ physicochemical parameters (Table S1). Furthermore, TPSA values are 150.15 for 3d and 167.22 Å2 for 3e. The results indicated that the calculated absorption percentages (ABS) for the synthesized derivatives were 57.19% and 57.19% respectively. This suggests that these derivatives may have the required bioavailability and ability to pass through cell membranes. Molecular 3d and 3e contained five and four rotatable bonds, respectively, which signifies their molecular adaptability with respect to their biotarget. One well-known factor that has a big impact on distribution and absorption characteristics is aqueous solubility. With hits of 16323.99 and 20701.67 mg/L (greater than 0.0001 mg/L), respectively, 3d and 3e met the solubility requirements. Similarly, human plasma protein binding (PPB), brain-blood barrier partition coefficient (BBB), human intestinal absorption, and Caco2 (colon adenocarcinoma) permeability coefficient were all evaluated in silico using Pre-ADMET software (Table S1).

In the Caco-2 cell model, 3d and 3e displayed medium cell permeability, with values ranging from 21.06 to 21.10 nm/s, respectively. The compounds were well-absorbed, as evidenced by the good human intestine absorption values (51.80–87.45%). Moreover, they failed to breach BBB. It was discovered that the two derivatives had modest binding rates (24.78–76.06%) to human plasma proteins. The objective of toxicity risk assessment is to identify chemical structure substructures that may indicate a toxicity risk in any of the three main toxicity classes: mutagenicity, irritancy, and mutagenicity. The mutagenicity, carcinogenicity, and tumorigenicity profiles of the chosen compounds were examined using the DataWarrior program; none of them showed any of these characteristics. We discovered that compound 3e was not a P-gp protein substrate (Table S1), suggesting that compound 3e had a very low probability of effluxing out of the cell and having a maximum effect. The Synthetic Accessibility (SA) Score, which is assigned by SwissADME, measures the ease of synthesis of a molecule. The grading scale is standardized and spans from 1 (representing the most challenging level of difficulty) to 10 (representing the least challenging level of difficulty) [29]. The large-scale synthesis of 3d and 3e was shown to be feasible, as evidenced by their respective SA scores of 2.83 and 3.11.

Docking into DNA gyrase A’s active site

The Protein Data Bank has made available the crystal structure of Gyrase A, which may be accessed using the accession code 4ckl. Docking attempts were conducted utilizing AutoDock Vina, a software application that requires the ligands and receptors to be in the form of pdbqt [32]. Upon redocking the complex enzyme with the co-crystallized ligand, the calculated root mean square deviation (RMSD) between the docked and co-crystallized ligands was 0.44 Ǻ (Fig. 6); thus, the docking method was validated. The findings were visually represented through the utilization of the Discovery Studio 4.5 visualizer [33]. Docking experiments were conducted on molecules 3d and 3e, which were identified as the most active in the active site of gyrase A.

Fig. 6
figure 6

Co-crystal component (Simocyclinone) superposed in its docked position

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DNA gyrase A is critical for preserving the natural topological configuration of DNA in the cell during the process of converting between relaxed and supercoiled forms [34]. The N-terminal domain of GyrA is composed of several critical amino acids, including Arg91, Met120, Val44, Lys42, His80, Gly81, Ser172, Ser97, and His45. These amino acids create a binding region for inhibitors and hinder the binding of DNA [35]. By employing a docking technique, the compounds 3d, 3e, and Simocyclinone D8 (SD8) were positioned within the binding domain of the E. coli gyrase A enzyme. The docking scores for these molecules were − 8.2, -8.3, and − 8.2 Kcal/mol, respectively. Simocyclinone established typical hydrogen bonds with Lys42, His 80, Arg91, Ser172, and Asn169. Conversely, it engaged in carbon-hydrogen bonding with His80 and His45, and also formed a hydrophobic interaction with His80. Additionally, it formed an Amide-Pi Stacked connection with Gly170 and Ser171, as depicted in Fig. 7, Table S2. The compounds are attached similarly to the N-terminal domain. The amide group of both compounds established three hydrogen bonds with Thr88, Arg91, and His45. Furthermore, compounds 3d and 3e exhibited a hydrophobic contact with Lys42, Val44, and Pro43, contributing to three or more interactions (Figs. 8 and 9, Table S2). In addition, compound 3d produced two additional hydrogen connections with Gly173 and His78. Compound 3e, alternatively, exhibited one extra hydrogen bond with Arg47. In addition, ciprofloxacin attached to the active site of gyrase A by establishing four hydrogen bonds with Val44 and Arg47, and it also makes six hydrophobic connections with His78, His80, and Pro43. The fluorine atom formed a halogen connection with Pro43, as shown in Fig. 10 and Table S2. The docking score of ciprofloxacin was − 7.7 kcal/mol.

Fig. 7
figure 7

3D and 2D representations of SD8 in the DNA gyrase A binding region

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Fig. 8
figure 8

2D and 3D representations of compound 3d in the DNA gyrase A binding region

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Fig. 9
figure 9

2D and 3D representations of compound 3e in the DNA gyrase A binding region

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Fig. 10
figure 10

2D and 3D representations of ciprofloxacin in the DNA gyrase A binding region

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Materials and methods

Chemistry

Instruments and Material (see supplementary file).

The data is available in the supplementary file.

General procedure for the synthesis of 1,2-diamino-5-cyano-6-oxo-4-(substituted phenyl)-1,6-dihydropyridine-3-carboxamide. (3a-e)

At a temperature of 80 °C, an equimolar solution of piperidine (20% mol), cyanoacetohydrazide 2 (1 mmol), cyanoacetamide 4 (1 mmol), and various aldehydes (1 mmol) was stirred within a solution of ethanol and H2O. After the reaction was completed, as monitored by thin-layer chromatography (TLC) using a 1:1 ratio of ethyl acetate to n-hexane, the solid product was filtered and then washed with a solution of water and ethanol (H2O/EtOH). This process resulted in the production of pure products 3a-e.

1,2-diamino-5-cyano-4-methyl-6-oxo-1,6-dihydropyridine-3-carboxamide (3a)

Yield 70%; m.p. 140 -142oC; IR (KBr, cm− 1): 3339, 3315, 3236 (3NH2), 3053 (CH aromatic),2989 (CH aliphatic), 2207 (C ≡ N), 1680, 1669(2 C = O), 1583 (C = C); 1H NMR (DMSO-d6)δ(ppm): 2.93 (s, 3 H, CH3), 5.86 (s, 2 H, NH2;exchangeable with D2O), 7.34 (s, 2 H, NH2; exchangeable with D2O), 7.69 (s, 2 H, NH2; exchangeable with D2O); 13C NMR (DMSO-d6) δ (ppm): 18.61, 91.18, 116.39, 117.41, 153.08, 157.15, 164.22, 174.59; MS, m/z: 207 (M+, 25.52%); Anal. Calcd. For C8H9N5O2 (207): C, 46.38; H, 4.38; N, 33.80,Found: C, 46.28; H, 4.40; N, 33.81.

1,2-diamino-5-cyano-4-(2,4-dichlorophenyl)-6-oxo-1,6-dihydropyridine-3-carboxamide (3b)

Yield 79%; m.p. 150 -152oC; IR (KBr, cm− 1): 3352, 3332, 3212 (3NH2), 3063 (CH aromatic), 2949 (CH aliphatic),2224 (C ≡ N), 1674, 1659 (2 C = O),1585 (C = C); 1H NMR (DMSO-d6) δ (ppm): 5.72 (s, 2 H, NH2; exchangeable with D2O), 7.44 (d, H, H6 Ar-H, J = 8 Hz), 7.65 (s, H, H3 Ar-H), 8.28 (d, 2 H, H5 Ar-H, J = 8 Hz), 8.47 (s, 2 H, NH2; exchangeable with D2O), 9.89 (s, 2 H, NH2; exchangeable with D2O); 13C NMR (DMSO-d6) δ (ppm): 95.35, 114.45, 126.55, 128.06, 131.26, 131.34, 133.53, 133.69, 134.48, 146.31, 160.00, 165.28, 169.61; MS, m/z: 337 (M+, 35.80%), 339 [(M + 2)+, 22.42%], 341 [(M + 4)+, 3.33%]; Anal. Calcd. For C13H9Cl2N5O2 (337): C, 46.18; H, 2.68; N, 20.71,Found: C, 46.08; H, 2.71; N, 20.68.

1,2-diamino-5-cyano-4-(4-(dimethylamino)phenyl)-6-oxo-1,6-dihydropyridine-3-carboxamide (3c)

Yield 80%; m.p. 145 -147oC;IR (KBr, cm− 1): 3396, 3332, 3252 (3NH2), 3022(CH aromatic),2922 (CH aliphatic),2208 (C ≡ N), 1678, 1665 (2 C = O), 1570 (C = C); 1H NMR (DMSO-d6) δ (ppm): 2.96 (s, 6 H, 2CH3), 6.68, (d, 2 H, H3,5Ar-H, J = 8 Hz), 7.43 (d, 2 H, H2,6Ar-H, J = 8 Hz), 7.63 (d, 4 H, 2NH2; exchangeable with D2O), 8.50 (s, 2 H, NH2; exchangeable with D2O); 13C NMR (DMSO-d6) δ (ppm): 43.74, 92.93, 110.41, 111.66, 111.71, 121.25, 121.55, 130.71, 152.00, 159.84, 161.93, 163.58; MS, m/z: 312 (M+, 42.52%); Anal. Calcd. For C15H16N6O2 (312): C, 57.68; H, 5.16; N, 26.91,Found: C, 47.58; H, 5.18; N, 26.92.

1,2-diamino-4-(4-(benzyloxy)phenyl)-5-cyano-6-oxo-1,6-dihydropyridine-3-carboxamide (3d)

Yield 63%; m.p. 90 -92oC;IR (KBr, cm− 1): 3321, 3332, 3185(3NH2),3002(CH aromatic),2933(CH aliphatic),2216(C ≡ N), 1677, 1669(2 C = O), 1585(C = C); 1H NMR (DMSO-d6) δ (ppm): 5.02 (d, 2 H, OCH2), 6.90 (d, 2 H, H3,5 Ar-H, J = 12 Hz), 7.04 (d, 2 H, H2,6 Ar-H, J = 12 Hz), 7.34–7.43 (m, 5 H, phenyl Hs), 7.82 (d, 4 H, 2NH2; exchangeable with D2O), 8.64 (s, 2 H, NH2; exchangeable with D2O); 13C NMR (DMSO-d6) δ (ppm): 69.20, 84.78, 113.35, 114.30, 114.89, 126.46, 127.80, 128.45, 129.05, 130.91, 137.09, 150.50, 157.03, 159.02, 169.22, 172.33; MS, m/z: 375 (M+, 13.65%); Anal. Calcd. For C20H17N5O3 (375): C, 63.99; H, 4.56; N, 18.66,Found: C, 63.97; H, 4.55; N, 18.67; HPLC: rt 8.12 min (purity > 95.0%).

4-(1,6-diamino-5-carbamoyl-3-cyano-2-oxo-1,2-dihydropyridin-4-yl)phenyl acetate (3e)

Yield 54%; m.p.280 -280oC; IR (KBr, cm− 1): 3334, 3313, 3201(3NH2), 3025(CH aromatic), 2987(CH aliphatic), 2208(C ≡ N), 1679, 1663(2 C = O), 1597(C = C); 1H NMR (DMSO-d6) δ (ppm): 2.50 (s, 3 H, CH3), 6.62, (d, 2 H, H3,5 Ar-H, J = 8 Hz), 7.18 (d, 2 H, H2,6 Ar-H, J = 8 Hz), 7.46 (s, 2 H, NH2; exchangeable with D2O), 8.42 (s, 2 H, NH2; exchangeable with D2O); 9.09 (s, 2 H, NH2;exchangeable with D2O); 13C NMR (DMSO-d6) δ (ppm): 23.94, 89.57, 113.63, 114.90, 126.26, 128.11, 129.88, 151.53, 155.92, 157.59, 157.44, 165.77, 172.72; MS, m/z: 327 (M+, 21.29%);Anal.Calcd. For C15H13N5O4 (327):C, 55.05;H,4.00; N, 21.40, Found: C, 55.06; H, 3.99; N, 21.42; HPLC: rt 8.11 min (purity > 95.0%).

General procedure for the synthesis of 1,2-diamino-5-cyano-6-oxo-4-(substituted phenyl)-1,6-dihydropyridine-3-carbothioamide. (4a-d)

The combination containing 1 mmol of cyanoacetohydrazide 2, 1 mmol of cyanothioacetamide 4, 1 mmol of aromatic aldehyde, and 20% mol of piperidine in a mixture of ethanol-water was agitated at 80 °C. Following the conclusion of the procedure, which was observed using thin-layer chromatography (TLC) with a mixture of ethyl acetate and n-hexane in a 1:1 ratio, the resulting product was obtained by filtering and subsequently rinsed with a mixture of water and ethanol (H2O/EtOH) to yield the pure products 4a-d.

1,2-diamino-5-cyano-4-methyl-6-oxo-1,6-dihydropyridine-3-carbothioamide (4a)

Yield 80%;m.p.230–232 oC; IR (KBr, cm− 1): 3344, 3320, 3230 (3NH2), 3021(CH aromatic), 2954(CH aliphatic), 2201(C ≡ N), 1665(C = O), 1587(C = C);; 1H NMR (DMSO-d6) δ(ppm): 2.51 (s, 3 H, CH3), 5.28 (s, 2 H, NH2; exchangeable with D2O), 6.24 (s, 2 H, NH2; exchangeable with D2O); 7.56 (s, 2 H, NH2; exchangeable with D2O); 13C NMR (DMSO-d6) δ (ppm): 18.56, 72.05, 115.55, 117.35, 143.70, 158.30, 161.46, 190.35; MS, m/z: 223 (M+, 11.69%); anal. Calcd. For C8H9N5OS(223): C, 43.04; H, 4.06; N, 31.37, S, 14.36, found: C, 43.06; H, 4.05; N, 31.40; S, 14.39

1,2-diamino-5-cyano-4-(2,4-dichlorophenyl)-6-oxo-1,6-dihydropyridine-3-carbothioamide (4b)

Yield 72%;m.p.204–206 oC;IR (KBr, cm− 1): 3364, 3342, 3208 (3NH2), 3019(CH aromatic), 2939(CH aliphatic), 2191(C ≡ N), 1653(C = O),1550(C = C); 1H NMR (DMSO-d6) δ (ppm): 7.53 (d, H, H6 Ar-H, J = 8 Hz), 7.69 (s, 2 H, NH2;exchangeable with D2O), 7.78 (s, H, H3 Ar-H), 8.11 (d, 2 H, H5 Ar-H, J = 8 Hz), 8.89 (s, 2 H, NH2;exchangeable with D2O), 10.27 (s, 2 H, NH2;exchangeable with D2O); 13C NMR (DMSO-d6) δ (ppm): 75.60, 116.30, 117.65, 125.25, 127.09, 128.13, 129.28, 133.40, 135.64, 145.73, 160.55, 165.22, 190.12; MS, m/z: 353 (M+, 20.23%), 355 ([(M + 2)+], 15.14%), 357 ([(M + 4)+], 2.04%); Anal. Calcd. For C13H9Cl2N5OS (353): C, 44.08; H, 2.56; N, 19.77;S, 9.05, Found: C, 44.10; H, 2.57;N, 19.15; S, 9.09.

1,2-diamino-5-cyano-4-(4-(dimethylamino)phenyl)-6-oxo-1,6-dihydropyridine-3-carbothioamide (4c)

Yield 80%;m.p. 260 -262oC; IR (KBr, cm− 1): 3392, 3352, 3199 (3NH2), 3082(CH aromatic), 2985(CH aliphatic), 2223(C ≡ N), 1673(C = O), 1557(C = C);1H NMR (DMSO-d6) δ (ppm): 3.04 (s, 6 H, 2CH3), 6.78 (d, 2 H, H3,5 Ar-H, J = 8 Hz), 7.63 (s, 2 H, NH2; exchangeable with D2O), 7.67 (d, 2 H, H2,6 Ar-H, J = 8 Hz), 8.49 (s, 2 H, NH2; exchangeable with D2O), 9.67 (s, 2 H, NH2; exchangeable with D2O);13C NMR (DMSO-d6) δ (ppm): 43.81, 73.65, 111.10, 111.71, 115.80, 124.53, 131.59, 145.73, 151.32, 163.43, 169.59, 189.93; MS, m/z: 328 (M+, 5.11%); Anal.Calcd. For C15H16N6OS (328): C,54.86; H,4.91; N, 25.59; S, 9.76, Found: C, 54.84; H, 2.57; N, 25.57; S, 9.73.

1,2-diamino-4-(4-(benzyloxy)phenyl)-5-cyano-6-oxo-1,6-dihydropyridine-3-carbothioamide (4d)

Yield 70%;m.p. 150–152oC; IR (KBr, cm− 1):3375, 3352, 3256 (3NH2), 3027(CH aromatic), 2999(CH aliphatic), 2209(C ≡ N), 1672(C = O), 1594(C = C); 1H NMR (DMSO-d6) δ (ppm): 5.18 (s, 2 H, OCH2), 7.20, (d, 2 H, H3,5Ar-H, J = 8 Hz), 7.34–7.48 (m, 5 H, phenyl Hs, 2 H, NH2; exchangeable with D2O), 7.86 (d, 2 H, H2,6Ar-H, J = 8 Hz), 8.63 (d, 2 H, NH2;exchangeable with D2O), 9.87 (s, 2 H, NH2;exchangeable with D2O); 13C NMR (DMSO-d6) δ (ppm): 69.33, 75.55, 114.19, 115.02, 115.21, 127.42, 128.07, 129.78, 131.80, 136.30, 142.12, 155.20, 160.48, 163.28, 169.90, 191.29; MS, m/z: 391 (M+, 11.59%); Anal.Calcd.For C20H17N5O2S (391):C,61.73;H,4.38;N,17.89; S,8.19, Found: C,61.70; H,4.36; N,17.91; S,8.20.

Biological evaluations

Antimicrobial, MIC, MBC, MFC assessment

In order to evaluate the effectiveness of newly produced molecules in preventing the growth of the studied microorganisms, an agar diffusion test was conducted on them. The analytical results were provided in respect to the dimensions of the produced inhibitory zones [36, 37]. MIC, MFC, and MBC assays were performed in accordance with the procedures outlined in the cited articles [19, 38, 39] (See the supplemental file).

DNA gyrase supercoiling assay [40]

(See supplementary file).

Time-kill curve calculation towards E. Coli [41]

(see supplementary file)

In vitro cytotoxicity assay [42]

(see supplementary file)

Conclusion

The primary objective of this investigation was to assess the antimicrobial effectiveness of carboxamide derivatives (3a-e) and carbothioamide compounds (4a-d). Most of the tested compounds exhibited a notably potent antibacterial action against the chosen Gram-positive bacteria in comparison to the standard reference drug. Moreover, the investigated gram-negative bacteria, particularly E. coli, experienced substantial inhibition from compounds 3b, 3d, 3e, 4b, and 4d. Furthermore, all evaluated analogs exhibited a satisfactory to moderate level of antifungal effectiveness. Compound 3d and compound 3e, which showed the most potential, were evaluated for their ability to impede DNA Gyrase A. Compound 3d exhibited significant inhibitory activity, with an IC50 value of 1.68 µg/mL. Moreover, the study of the kinetics at which compounds 3d and 3e eliminate bacteria showed that they exhibit bactericidal activity within 90–120 min of being exposed to E. coli. In addition, compounds 3d and 3e exhibited favorable pharmacokinetic characteristics. Furthermore, the docking studies revealed a robust affinity for the DNA Gyrase A binding site.

Data availability

Data is provided within the manuscript or supplementary information files.

Abbreviations

MIC:

Minimum Inhibitory Concentrations

E.coli:

Escherichia coli

P. aeruginosa:

Pseudomonas aeruginosa

C. albicans:

Candida albicans

1H NMR:

Proton Nuclear Magnetic Resonance

13C NMR:

Carbon-13 nuclear magnetic resonance

IR:

Infrared

B. pumilus:

Bacillus pumilus

S. faecalis:

Streptococcus faecalis

S. cerevisiae:

Saccharomyces cerevisiae

E. cloacae:

Enterobacter cloacae

IZ:

Inhibition Zone

MBC:

Minimum Bactericidal Concentration

MFC:

Minimum Fungicidal Concentration

SAR:

Structure-Activity Relationship

TPSA:

Topological Polar Surface Area

BBB:

Brain-Blood Barrier

PPB:

Plasma Protein Binding

SA:

Synthetic Accessibility

RMSD:

Root Mean Square Deviation

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Acknowledgements

Acknowledgement to Deanship of Scientific Research and Libraries at Princess Nourah bint Abdulrahman University for funding Grant No. (FRP-1445-4)

Funding

This research was funded by the Deanship of Scientific Research and Libraries at Princess. Nourah bint Abdulrahman University, through the Research Funding Program, Grant No. (FRP-1445-4)

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Authors and Affiliations

  1. Department of Pharmaceutical Sciences, College of Pharmacy, Princess Nourah Bint Abdulrahman University, P.O. Box 84428, Riyadh, 11671, Saudi Arabia

    Omkulthom Al Kamaly & Aisha A. Alsfouk

  2. Department of Pharmaceutical Medicinal Chemistry and Drug Design, Faculty of Pharmacy (Girls), Al-Azhar University, Cairo, Egypt

    Amel S. Younes, Marwa F. Harras, Rehab Sabour & Mona H. Ibrahim

Authors
  1. Omkulthom Al Kamaly
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  2. Amel S. Younes
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  3. Marwa F. Harras
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  5. Aisha A. Alsfouk
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  6. Mona H. Ibrahim
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Contributions

Conceptualization: M.H.I, M.F.H, R.S. Methodology, M.H.I, A.S.Y. Software, M.H.I, A.S.Y. A.A.A.Validation, O.A.K, A.A.A, M.F.H, Formal analysis, M.H.I, M.F.H, R.S. A.S.Y. O.A.K, A.A.A. Investigation M.H.I, M.F.H, R.S. A.S.Y. Data curation, M.H.I, A.S.Y. M.F.H. Writing—original draft preparation, M.H.I, M.F.H, A.S.Y. Writing—review and editing, M.H.I, M.F.H, R.S. A.S.Y, O.A.K, A.A.A. Visualization, M.H.I, M.F.H, O.A.K. Funding acquisition, O.A.K, A.A.A.

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Correspondence to Mona H. Ibrahim.

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Kamaly, O.A., Younes, A.S., Harras, M.F. et al. New N-amino-5-cyano-6-pyridones as antimicrobial small molecules endowed with DNA gyrase a inhibitory activity: design, one-pot synthesis, biological assessment and in silico insights. BMC Chemistry 18, 226 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13065-024-01342-9

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BMC Chemistry

ISSN: 2661-801X

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