Synthesis, antibacterial evaluation, in silico ADMET and molecular docking studies of new N-acylhydrazone derivatives from acridone

2.1 Synthesis

The synthesis of novel acylhydrazones based on acridone compounds was depicted in Scheme 1. Initially acridone was reacted with ethyl 2-bromoacetate using potassium carbonate and Tetra butylammonium bromide (TBAB) as phase transfer catalyst in DMF at 70 °C. The treatment of the ethyl ester (1) with hydrazine afforded the compound (2). The condensation reaction of the compound (2) with variously substituted aldehydes and ketone in ethanol give the expected new acylhydrazones 3a-k in good yields.

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Scheme 1 Synthesis route of novel N-acylhydrazone derivatives from acridone (3a-k).

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The structure of these compounds was ascertained by ¹H NMR, ¹³C NMR, IR and mass spectral data. The IR spectra of compounds (3a-k) showed characteristic absorption bands in the region of 1600–1611 cm⁻¹ corresponding to the vibration of the imine function (C⚌N), and two bands corresponding to the stretching vibrations of the carbonyls of the acridone ring and the hydrazone function in the region of 1638 and 1680 cm⁻¹, respectively. However the desperation of the vibration bonds of amine (NH₂) group in the region of 3226 cm⁻¹ confirmed the formation of compounds.

¹H NMR spectra of the compounds (3a-h) in DMSO‑d₆, gave two sets of resonance signals which can be attributed to the existence of conformational isomers in DMSO‑d₆ (E,trans and E,cis) (Fig. 1). The splitting of signals were observed for amide (CONH) between 12.10 and 11.77 ppm, methylene (NCH2) to 5.80–5.17 ppm and imine between 8.50 and 8.07 ppm (N⚌CH). In the compound 3k, the isomerization around the double bond (C⚌N) is degenerated. Thus, the duplication of signals to 5.59–5.36 ppm and 10.77–10.66 ppm in the ¹H NMR spectrum can be attributed to the existence of cis/trans-amide conformers only. In ¹³C NMR, the spectra also show two sets of signals to 47.11–48.50 ppm and 168.94–163.38 ppm corresponding to the signal of the methylene (—N—CH₂—) and amide (—NHCO), respectively, while the signal of the imine (C⚌N) to 149.85–145.30 ppm appears as a single signal.

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Fig. 1 E,trans and E,cis conformations in acylhydrazone derivatives.

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The ¹H NMR spectrum of the compound 3a shows two signals at 5.80 and 5.35 ppm attributable to the protons of the methylene (NCH₂), the signal at 5.80 ppm corresponds to the E, trans conformation with 73% abundance and the signal at 5.35 ppm assigned to the E, cis conformation with an abundance of 27%. Approximately the same abundance is found in the case of amide proton (CONH) at δ 12.01 ppm (23%, E, cis conformation) and 11.97 ppm (77%, E, trans conformation) and for the imine proton (N⚌CH) signals at δ 8.33 ppm (25%, E, cis conformation) and δ 8.18 ppm (75%, E, trans conformation). The signal intensities of the methylene, imine and amide protons allowed us to measure the ratio of cis/trans-amide conformers of 3a-h, the percentage of cis/trans conformers in DMSO‑d₆ are summarized in Table 1.

2.2 Antibacterial activity

The novel acylhydrazone (3a-k) were tested for their in vitro antibacterial activities against the human pathogens three gram negative bacteria Pseudomonas putida, Klebsiella pneumonia and Escherichia coli and one gram positive bacteria Staphyloccocus aureus. The purity of compounds (3a-k) was confirmed by HPLC.

As revealed from Table 2, the compounds 3a, 3b and 3e were found to have good antibacterial activity against Staphylococcus aureus, the compound 3a was found to be the best active derivatives with MIC = 19.61 µg/mL. Moreover, the compound 3f showed high antibacterial potential against Pseudomonas putida (38.46 µg/mL) which is very close to that obtained with the commercial antibiotic Chloramphenicol (37.03 µg/mL). The tested compounds 3a-k shown moderate activity against Escherichia coli with the MIC values between 38.46 and 74.0 μg/mL. The low antibacterial activity was observed against Klebsiella pneumonia. The results indicate that the substitution of the acridone ring with acylhydrazone increase the antibacterial activity against all bacteria. Moreover the N-acylhydrazone acridone derivatives 3a-h containing a secondary aldimine fragment showed interesting antibacterial activity compared with the 3k derivative containing a secondary ketimine.

2.3 In silico studies

To better, understand as well as to support the in vitro antibacterial activity of the synthesized compounds for the rational design of new potent molecules. In this manuscript molecular docking studies of the most active compounds have been made to study the interactions and clarify the probable binding modes between acridone derivatives and DNA gyrase complex (PDB ID : 3TTZ) of Staphyloccocus aureus, and transcriptional regulator (TtgR) enzyme (PDB ID : 2UXI) of pseudomonas putida, which provide straightforward knowledge for further structural optimization.

2.4 Docking for DNA-gyrase of Staphyloccocus aureus

The docking studies were carried out using the bacterial DNA gyrase complex obtained from Protein Data Base (PDB ID 3TTZ) (Sherer et al., 2011). Validation of the docking process was done by redocking of the co-crystalized ligand (07 N) to the ATP binding site of DNA gyrase and RMS (Root Mean Square) distance between the docked and the experimental co-crystallized binding pose was only 1.63 Å (less than 2 Å), which is satisfactory. Fig. S31 shows that the docked structure (magenta color) and the X-ray crystal structure (green color) are quite similar. In addition, all the eight acridones were docked into the binding pocket of DNA gyrase enzyme successfully. The molecular docking representation for each synthetic compound and the superposition of all best docking pose in the enzyme binding pocket are shown in Figs. S32-S33 (supplementary data).

Surflex-dock module from Sybyl-X 2.0 software (Tripos International, 2012) was used to predict the proposed binding mode, binding affinity, preferred orientation of each docking pose of the synthesized compounds with DNA gyrase structure. The calculated interaction energies for the synthesized compounds were in agreement with experimental result which showed that 3a, 3b, 3c, 3d, 3e, 3f, 3g and 3h are potent inhibitors of DNA-gyrase as compared to the other members and reference standard.

It has been revealed from binding studies that all the synthesized compounds 3a-3h fit snugly making various close contacts with the residues lining the active site of DNA gyrase.

It presented important interactions with the DNA-gyrase complex. The acridone moiety was involved in aromatic stacking interactions with Pro87 and Pi-cation with Arg84. The NH group of the amide group formed a hydrogen bond with the Thr173. Also, the benzene aromatic ring was involved in Pi-Alkyl interaction with Ile86. Hence, docking studies revealed the strong binding affinity of 3a at the ATP binding site of GyrB, which may be responsible for its significant in vitro antibacterial activity especially against S. aureus.

Compound 3e showed a binding affinity (-log ki) value of 6.16. The acridone moiety was involved in Pi-Anion and Pi-Cation interactions with Glu58 and Arg84. The NH of the amide group formed a hydrogen bond with Asn54. Moreover, the carbonyl of the acridone moiety formed a hydrogen bond with Gly85.

The proposed binding mode of compound 3b showed a binding affinity value of 6.15. The acridone moiety was involved in hydrophobic interactions with Ile85, Ile102 and Ile175. Its carbonyl of the amide group formed a hydrogen bond with Thr173. In addition, the benzene ring showed a Pi-Alkyl interaction with Pro87.

From the different interactions of the synthesized compounds in Fig. 2, Table 3 and Figs. S32-S39, it can be concluded from the docking results that the eight compounds had H-bond interactions with the receptor indicating that the H-bond interactions play an important role of the inhibition of the DNA-gyrase. Additionally, they all bind to ATP binding site of DNA gyrase and share largely homogeneous in binding mode (especially hydrogen bond with Asn54, Arg144 and Thr173) to several DNA-gyrase inhibitors reported in the literature (Alves et al., 2014; Baig et al., 2015), Therefore, that can prove our docking process was reasonable. While the main cause of their mediated antibacterial activity is considered due to those compounds are aligned adequately within the ATP binding site of DNA-gyrase, which allows them to make important interactions with this pocket. The results of antibacterial activity are supported by docking analysis. The high docking scores and binding pattern of compounds of 3a, 3b, 3c, 3d, 3e, 3f, 3g and 3h, in Fig. 2, Table 3 and Figs. S32-S39, reveal that these compounds are well accommodated in active site of enzyme and they strongly interact within the active site of DNA gyrase enzyme (3TTZ). The molecules 3b, 3c, 3d, 3f, 3g and 3h were found to be orienting in opposite direction of compounds 3a and 3e as shown in supporting information Figs. S32-39.

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Fig. 2 Binding mode of compound 3a with DNA-gyrase complex, the hydrogen bonds are presented in green dashed lines.

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2.5 Docking for transcriptional regulator (TtgR) enzyme of pseudomonas putida

For pseudomonas putida antibacterial activity the docking studies were carried out using the bacterial the transcriptional regulator (TtgR) enzyme (PDB Code: 2UXI) (Alguel et al., 2007) obtained from Protein Data Base.

In order to validate the docking approach, the co-crystallized ligand (Phloretin) was docked to the active site of the studied enzyme and RMS (Root Mean Square) distance was calculated, which was found 0.70 Å. (less than 2 Å), which is satisfactory. Fig. S40 shows that the docked structure (magenta color) and the X-ray crystal structure (green color) are quite similar. In addition, all the eight acridones were docked into the binding pocket of DNA gyrase enzyme successfully. The molecular docking representation for each synthetic compound and the superposition of all best docking pose in the enzyme binding pocket are shown in Figs. S41-S48 (supplementary data).

The Surflex-dock module was used to predict the proposed binding mode, affinity, preferred orientation of each docking pose and binding affinity of the synthesized compounds with transcriptional regulator (TtgR) enzyme. The calculated interaction energies for the synthesized compounds were in agreement with experimental result which showed that 3a, 3b, 3c, 3d, 3e, 3f, 3g and 3h are potent inhibitors of transcriptional regulator (TtgR) enzyme as compared to the other members and reference standard.

From the analysis of the active site of TtgR, it was observed that the residues Cys 137 and Asn110 along with Ala 74, Ser 77, Glu 78, Val 96, Ile 175 and VAL171 seem to play crucial role in binding with the ligands and triggering the efflux pump. The residues Asn 10 and Cys 137 probably are the most instrumental in binding through strong H-bonding and sensing the compounds toxic to the organism (Table 4 and Fig. 3).

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Fig. 3 Binding mode of compound most active compound 3f with TtgR enzyme.

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The residues Ala 74, Ser 77, Glu 78, Val 96, Ile 175 and Val 171 interact with the ligands mostly by hydrophobic interactions with the ligands as shown in Table 4 and Figs. S41-S48.

As it has been observed that, the antimicrobials phloretin, naringenin, and quercetin bind strongly with those residues (Alguel et al., 2007).

2.6 ADMET

The ADMET parameters of the newly synthesized compounds (3a-h) were evaluated using the pkCSM (Pires et al., 2015) and AdmetLab (Ison et al., 2016) online tools. For a given compound a logBB < −1 considered poorly distributed to the brain. Positive result in AMES test suggests that compound could be mutagenic. Prediction of ADMET parameters are listed in Table 5.

Pharmacodynamics results indicated that compounds (3a-h) may prove good lead as antibacterial drugs by targeting DNA gyrase and transcriptional regulator (TtgR) enzyme. For drug formulation pharmacokinetic parameters are likewise a significant prospect and should be brought into thoughtfulness. The major properties studied for different synthesized compounds (3a-h) were to be acceptable and were incorporated in Table 5. The % oral intestinal drug absorption predicted for all compounds 3a-h was highly satisfactory with a high percentage (>90%), indicating their possibilities in oral drug formulation for the treatment of staphyloccocu aureus and pseudomonas putida infections. For the brain/blood partition coefficient of the compounds (3a-h) was predicted, thus, newly synthesized compounds presented little chance to cross the blood–brain barrier. In addition, rapid renal clearance is associated with small and hydrophilic compounds. The evaluation of the toxicity profile for compounds (3a-h) including the human ether-a-go-go related gene (hERG), AMES test, Max. Tolerated dose (human), Skin sensitization and Minnow toxicity indicate that acylhydrazones 3a-h apparently do not have potential toxicity. Results of ADMET and bioavailability (Rule of Five) (Lipinski, 2004) as depicted in Tables 5 and 6, were within the acceptable limits for the designed compounds in both lipophilicity and solubility.

According to the in vivo pharmacodynamics results and in silico ADMET parameters, the newly synthetized compounds showed very interesting properties, in terms of intestinal adsorption, toxicity and Blood-brain barrier permeability; thus, it can be concluded that compounds 3a and 3f present good antibacterial activities against staphyloccocu aureus and pseudomonas putida infections, respectively. In addition, the present good drug-like characteristics and in silico ADMET parameters, consequently, they have more favorable properties as such and on further lead optimization may guide to novel compounds against staphyloccocu aureus and pseudomonas putida infections.

4.1 Materials

All materials were purchased from commercial suppliers. The ¹H, ¹³C NMR spectra was recorded with Bruker Avance 300. Mass spectrometric measurements were recorded using Exactive™ Plus Orbitrap Mass Spectrometer.IR spectra were recorded using JASCO FT-IR 4100 spectrophotometer.

4.2 Antibacterial activity

The novel acylhydrazones (3a-k) were evaluated for their in vitro antibacterial activity by the disk diffusion method, the active compounds were subjected to the determination of the MIC, using the broth Microdilution method. The microorganisms utilized for the test were Escherichia coli, Staphylococcus aureus, Pseudomonas putida and Klebsiella pneumonia. They were collected from clinical isolates. Bacterial inoculums were prepared by subculturing microorganisms into MHB at 37 °C for 18 h and were diluted to approximately 10⁶ CFU mL⁻¹. Initial solution with concentration 0.5 mg/mLof the compounds (3a-k) were prepared in DMF, further serial dilutions were made in the microplates and 100 μL of MHB containing each test microorganism were added to the microplate (Holetz et al., 2002; Olson et al., 2005; Sarkar et al., 2007), then incubated at 36 °C for 24 h. After incubation, 20 μL of TTC (0.04 mg/mL) were added to each microplate. The Color changes of TTC from colorless to red were accepted as microbial growth (Upadhya et al., 2013).

4.3 Molecular docking studies

Molecular docking of the compounds (3a-h) into the S. aureus DNA gyrase complex structure (PDB ID 3TTZ) (Sherer et al., 2011) and the transcriptional regulator (TtgR) enzyme of p. putida (PDB Code: 2UXI) (Alguel et al., 2007) was carried out using the SYBYL-X 2.0 molecular modeling package. The structures of 3TTZ and 2UXI in docking studies were downloaded from PDB. For protein preparation, the hydrogen atoms were added, water and impurities were removed. Different types of interactions between protein and the docked compounds (3a-h) were analyzed using Discovery Studio (Please, see the attached supplementary information).

4.4 Synthesis