Synthesis, characterization, in silico molecular docking, and antibacterial activities of some new nitrogen-heterocyclic analogues based on a p-phenolic unit

Nitrogen-containing heterocycles have shown pharmacological properties against various diseases. Herein, in our study, flavoHB enzyme is a highly promising well-validated target for identification of antibacterial inhibitors using in silico and in vitro techniques. To identify a new class of antimicrobial agents, N-(4-hydroxyphenyl)-3-oxobutanamide was utilized as a precursor in the synthesis of several nitrogen-based heterocycles (pyridine, pyrimidine, and pyrazole) attached to p-phenolic substrates 2–8. Treatment of 3-oxobutanimide 1 with malononitrile and/or ethyl cyanoacetate in ethanolic piperidine afforded the pyridinone analogues 2a,b. On the other hand, treatment of 1 with arylidene cyanothioacetamide furnished the pyridinthione derivative 3. The reaction of starting material 1 with salicylaldehyde and/or dimethyl formamide dimethyl acetal (DMF-DMA) yielded the pyridinones 4 and 5, respectively. Reaction of 1 with terephthalaldehyde and urea or thiourea gave bis structures 6a,b. The reaction of compound 1 with ethyl isothiocyanate and hydrazine hydrate afforded pyrimidine and pyrazole derivatives 7 and 8, respectively. The structures of newly prepared compounds 2–8 were elucidated using elemental data and spectral analyses such as IR, 1H NMR, 13C NMR, and MS. In addition, an in-house nitrogen-containing heterocycle analogues library 2–8 was examined and screened in vitro for their antibacterial effects against Gram-negative bacteria, Escherichia coli and Gram-positive bacteria, Staphylococcus haemolyticus, Kocuria kristinae, Enterococcus casseliflavus, and Bacillus cereus. Compounds 6a and 6b have also shown the highest antibacterial activity against all types of bacteria strains tested except Kocuria kristinae. Further, the molecular docking study of the newly prepared compounds with the target enzyme flavohemoglobin (flavoHB) was undertaken to explore their potential inhibitory activities. The results of the docking study indicated that compounds 6a and 6b have exerted the highest docking scores against the active site of flavoHB. As a result, the in vitro and molecular docking study findings suggested that the compounds 6a and 6b (with pyrimidine moiety, amide linkage, and phenolic substrate) might be potent bacterial flavohemoglobin (flavoHB) inhibitors and they could set a promising starting point for future design of antibacterial agents.


Introduction
Nitrogen-containing heterocycles 1-3 such as pyridine, pyrimidine, and pyrazole are important molecular building blocks that are involved in the structural composition of crucial chemicals for humans, including pharmaceuticals, natural resources, veterinary and agricultural products, analytical reagents, and dyes. [4][5][6] In addition, nitrogen-based heterocycles attached to phenolic substrates have received increasing attention due to their pharmacological properties including anticancer, antimalarial, antimicrobial, and antitubercular 6 activities. The pursuit of these properties requires efficient synthetic routes that allow rapid construction of diverse aromatic heterocycles with dened substitution patterns. Moreover, N-heterocyclic compounds coupled with the phenolic substrate possess a wide range of medicinal and pharmaceutical activities and are constituents of bioactive molecules such as antibiotics, vitamins, and pharmaceutics, as represented in Fig. 1. As aforementioned, there are three pharmacophoric features for good antibacterial activity including an N-heterocyclic system, a linker containing a functional group (amide), and an OH group which forms hydrogen bonds, as shown in Fig. 2.
On the other hand, Helmick et al. reported that nitrogencontaining heterocycles such as azoles had antibacterial activity through inhibition of a bacterial heme-containing enzyme called avohemoglobin (avoHB). 7 The avohemoglobin is a nitric oxide dioxygenase that used for oxidation of nitric oxide to nitrate. 8 The function of avohemoglobin is pivotal for the bacterial survival by protecting bacteria from nitrosative stress. 9 Thus, avoHB enzyme has been selected as therapeutic target for identication of antibacterial drug candidates.
Moreover, the molecular docking studies [26][27][28][29][30] of the newly prepared compounds with the target enzyme avohemoglobin (avoHB) were conducted to explore the potential inhibitory activity of these compounds. Finally, ADMET (absorption, distribution, metabolism, excretion, and toxicity) and drug-  likeness predictions of the compounds were calculated using Molinspiration, SwissADME and admetSAR tools. The compounds 6a and 6b (with pyrimidine moiety, amide linkage, and phenolic substrate) showed the strongest antibacterial activities, and exhibited the highest binding affinity to the target enzyme avohemoglobin (avoHB) with binding energies DGs (À10.4 and À10.0 kcal mol À1 ), respectively, higher than of the standard drug.

Chemistry
In the current work, treatment of 3-oxobutanamide 1 with active methylene compounds like malononitrile and ethyl cyanoacetate was achieved as represented in Fig. 3. So, treatment of 1 with malononitrile in ethanolic piperidine solution afforded the pyridinone derivative 2a. IR spectrum of the desired 2a showed disappearing of acetyl carbonyl and appearance one CN at n 2194 cm À1 . 1 H NMR of the structure showed singlet signal at d 2.40 ppm assigned to CH 3 , singlet signal for NH 2 group appeared at d 3.73 ppm, singlet signal corresponding to CHpyridine at d 7.64 ppm, and OH showed as hump at d 9.33 ppm. Similarly, the reaction of 1 with ethyl cyanoacetate instead of malononitrile, the reaction takes the way itself not on the ester as expected to give compound 2b. The IR spectrum of 2b showed the presence of ester carbonyl band at n 1721 cm À1 , amide carbonyl at n 1635 cm À1 and disappearance of CN absorption band. Moreover, 1 H-NMR spectrum revealed a triplet and quartet signals at d 1.12 and 4.04 ppm for CH 3 and CH 2 groups of esters, respectively. In addition, the other CH 3 group appeared as a singlet at d 2.28 ppm and appearance of the NH 2 protons at d 5.14 ppm as a singlet, in addition to CHpyridine at d 8.12 ppm in interference with the aromatic protons, as well as a singlet signal for OH proton appeared at d 10.32 ppm.
On the other hand, treatment of compound 1 with benzylidene cyanothioacetamide namely, 2-cyano-3-phenylprop-2enethioamide furnished the pyridinthione derivative 3, as shown in Fig. 4. Its IR spectrum showed band at n 2216 cm À1  assigned to CN group, and amide carbonyl at n 1631 cm À1 . In addition, 1 H NMR spectrum of compound 3 revealed a singlet signal at d 1.66 ppm assigned to CH 3 , singlet signal at d 3.93 ppm assigned to OH group, NH appeared at d 9.46 ppm and other NH appeared at d 9.83 ppm.
The reaction of 1 with salicylaldehyde takes the same way too, where the formyl group attacks the active methylene of the starting material to furnish the pyridinone 4, as shown in Fig. 5. The IR spectrum showed ester carbonyl at n 1707 cm À1 and amide carbonyl at n 1662 cm À1 . Moreover, 1 H NMR spectrum showed singlet signal at d 2.38 ppm assigned to CH 3 , d 8.88 ppm CH-pyridine appeared and hydroxyl group appeared at d 9.55 ppm.
During this phase of our research, we have shown that 3oxobutanamide 1 is expected to react with dimethylformamidedimethyl acetal (DMF-DMA) in reuxing dry xylene to yield the product which may be enaminone (i) as literature procedure. 31 But under the reaction conditions we obtained only a product that could be formulated as 5-acetyl-N,1-bis(4-hydroxyphenyl)-4methyl-6-oxo-1,6-dihydropyridine-3-carboxamide 5. However, the spectral data and chemical evidence did not t this structure. So, the structure (i) was readily ruled out as nal product, but it was formed as intermediate based on spectral data. The mass spectrum showed the molecular ion peak at m/z ¼ 377 (M + ) corresponding to molecular formula (C 21 H 18 N 2 O 5 ). The IR spectrum of compound 5 showed acetyl carbonyl at n 1710 cm À1 , and amide carbonyl at n 1650 cm À1 . 1 H NMR spectrum revealed singlet signal at d 1.91 ppm referred to acetyl CH 3 , singlet signal at d 2.18 ppm for other CH 3 , CH-pyridine noted at d 8.21 ppm, OH appeared as hump at d 9.50 ppm, and NH appeared at d 11.46 ppm. Moreover, 13 C NMR spectrum revealed two singlet signals assigned for two CH 3 at d ¼ 19.73 and 21.47 ppm, three singlet signals assigned for carbonyls at d 164. 25, 176.37, 196.46 ppm as declared in Fig. 6.
The reaction of 1 with a mixture of terephthaldehyde and urea or thiourea afforded the bis-structure 6a,b which conrmed on the bases of its compatible spectroscopic data as represented in Fig. 7. IR spectrum of 6a showed two amide carbonyls at n 1662 cm À1 . 1 H NMR spectrum showed singlet signal at d 2.28 ppm assigned to CH 3 , singlet signal at d 5.14 ppm assigned to OH group, singlet signal at d 9.61 assigned to NH, and other NH appeared at d 10.32 ppm. 13 C NMR showed singlet signal at d ¼ 17.62 ppm assigned to CH 3 group and singlet signal at d ¼ 165.57 ppm assigned to carbonyl group in addition to other carbons in the molecule. Similarly, IR spectrum of compound 6b showed band at n 1686 cm À1 for amide carbonyl group. 1 H NMR spectrum showed singlet signal at d 2.33 ppm assigned to CH 3 group, singlet signal at d 5.29 ppm for OH group, NH amide appeared at d 8.12 ppm, and NH pyrimidine appeared at d 10.00 ppm. 13 C NMR declared carbonyl at d ¼ 175 ppm and C]S at 192 ppm, and DEPT 135 revealed disappeared of CH 2 signal. Mass spectrum showed molecular ion peak at 595 m/z corresponding to M À1 molecular formula C 30 H 24 N 6 O 4 S 2 .
Also, the reaction of 3-oxobutanamide 1 with ethyl isothiocyanate afforded the pyrimidine derivative 7 via attaching rstly on amidic NH not on methylene as expected (Fig. 8). Structure 7 was conrmed by compatible spectroscopic data and elemental analysis. 1 H NMR spectrum of compound 7 showed singlet signal at d ¼ 1.10 ppm assigned to CH 3 , aromatic protons appeared at d 6.89-7.27 ppm as doublet of doublet signals, CH-pyrimidine appeared at 9.32 ppm, and broad signals appeared at d 9.77 and 10.15 ppm referred to SH, and OH groups, respectively. 13 C NMR conrmed the presences of CH 3 signal at d ¼ 14.82 ppm, and one carbonyl at d ¼ 165.72 ppm, in addition to other carbons in the structure. Moreover, mass spectrum showed the molecular ion peak at 236 m/z corresponding to molecular formula C 11 H 12 N 2 O 2 S.
On the other hand, 3-oxobutanamide 1 was treated with hydrazine hydrate to give the pyrazole derivative 8 which conrmed ay compatible spectroscopic data and elemental analysis, as declared in Fig. 9. The suggested mechanism for preparation of compound 8 is shown in Fig. 10. The IR spectrum of compound 8 revealed disappearance of two carbonyls. 1 H NMR spectrum showed singlet signal at d ¼ 2.10 ppm assigned to CH 3 , singlet signal referred to CH 2 at d ¼ 2.37 ppm, OH group appeared at d ¼ 5.23 ppm, NH appeared at d ¼ 5.81 ppm, and doublet of doublet signals at d ¼ 6.43, and 6.51 ppm referred to four aromatic protons. All spectral data of the prepared compounds are represented in ESI Section as Fig. S1-S21. †

Antibacterial assay
A disc diffusion method (Kirby-Bauer test) 32 was used to determine the antimicrobial activity of the newly prepared compounds 2-8. Overnight bacterial cultures were diluted to 1 : 10.000 into tryptic soy broth (TSB), by measuring and adjusting the bacterial suspension OD 595 , using spectrophotometer, Spectronic™. Nutrient agar (OXOID) for bacteria was inoculated with microbial cell suspension (200 mL in 20 mL medium), and poured into sterile Petri dishes. Sterile paper discs of 6 mm diameter saturated with tested compound placed on the surface of the inoculated agar plates, the susceptibility testing following the guidelines of the National Committee for Clinical Laboratory Standards. 33,34 Negative control was done using paper discs loaded with 20 ml of distilled water. Incubate overnight (24 h) at 37 C. At the end of the incubation period the antibacterial activity was evaluated by measuring the inhibition zones. 35 The result presented in Table 1, showed that compounds 6a and 6b had the best antibacterial activities close to standard drug against all types of bacteria strains tested except Kocuria kristinae. The compounds 4 and 7 had moderate antibacterial activity against Gram-negative bacteria Staphylococcus haemolyticus. Finally, compounds 2a,b, 3, 5, and 8 had not any antibacterial activities against any tested bacterial strains ( Table 1). The antibacterial activity is shown in the ESI Section, as Fig. S22. † MIC values showed promising results for most tested compounds as shown in Table 2. Within the tested bacteria, the MIC values were ranged from 0.5 to 2 mg mL À1 . Interestingly, both Gram-negative and Gram-positive bacteria were sensitive to the compounds. The antimicrobial activity can be modulated by the presence of pyrimidine moiety, amide linkage, and phenolic substrate.

Computational study
To investigate the affinity and orientation of the newly synthesized molecules against the active site of the target enzyme, an in silico study was performed. 36,37 In the present study, bacterial avoHB enzyme (PDB: 3OZU) 38 has been selected as therapeutic target for identication of antibacterial drug candidates   through molecular docking technique. In addition, levooxacin was selected as a standard drug to compare its binding affinity with those of the docked compounds.
The compounds 6a,b with the strong antibacterial activities, exhibited the highest binding affinity to the target enzyme with binding energies DGs (À10.4 and À10.0 kcal mol À1 ),   (Table 2). On the other hand, the standard drug, levooxacin exhibited binding energy of À7.9 kcal mol À1 , and showed two HBs and one arene-arene interactions with the residues Tyr393 and Glu388 at the distances 3.18, 2.40, and 4.55Å, respectively ( Table 3).
The 2D and 3D representations of the intermolecular interactions between all docked compounds and standard drug with the active site residues of the target enzyme avoHB are shown in Fig. 11. Furthermore, the ADMET and drug-likeness results showed interesting values for the compounds (Table 4). All the prepared compounds have molecular weights in the expected range of 130-725 g mol À1 . In addition, they have better human intestinal absorption (HIA + ) score in the range of 66.31-100%, which veried that they could be better absorbed by the human intestine. Further, HBA (H-bond acceptors) and HBD (H-bond donors) satisfy Lipinski's rule of ve and were found to be in the acceptable range. The rotatable bonds in the compounds are in limits (1-6). All ligand molecules except 4 and 8 does not pass the blood-brain barrier that conrming their good CNS safety prole. Finally, all the compounds displayed negative carcinogenicity tests. According to Lipinski's rule of ve which states that a molecule violating more than one, it is less likely to be a drug. Therefore, the ndings conrmed that the ligand molecules 6a and 6b that containing pyrimidine moiety, amide linkage, and phenolic substrate could be used as potent and safe avoHB inhibitors as promising antibacterial therapeutics.

Chemistry
All melting points were determined on an electrothermal apparatus and are uncorrected. IR spectra were recorded (KBr discs) on a Shimadzu FT-IR 8201 PC spectrophotometer. 1 H-NMR and 13 C-NMR spectra (400 MHz) were recorded in DMSO (CD3) 2 SO solution on a BRUKER500 FT-NMR system spectrometer, and chemical shis are expressed in ppm units using TMS as an internal reference. Mass spectrum was recorded on a GC-MS QP1000 EX Shimadzu. Elemental analyses were carried out at the Microanalytical Center of Cairo University, Egypt.
General procedure for preparation of compounds 2a,b. A mixture of compound 1 (1.93 g, 10 mmol) and malononitrile (0.66 g, 10 mmol) or ethyl cyanoacetate (1.13 g, 10 mmol) was reuxed in ethanol containing few drops of piperidine for 5 h. The reaction mixture was le to cool, then poured onto ice/ water containing few drops of conc. HCl. The formed solid product was collected by ltration and recrystallized from ethanol to afford pyridinone derivative 2a,b.

Determination of the minimum inhibitory concentration (MIC)
The determination of MIC was assayed as described earlier by Salem et al. 39 The freshly prepared cultures of both Gramnegative bacteria and/or Gram-positive bacteria were adjusted to OD 595 of 0.001. 100 mL of each isolate culture was put into sterilized 96-well plates. Then 20 mL of the screened compounds (2 mg mL À1 ) (serial dilutions of 10 À1 to 10 À10 were used, 8 replicates were made for each dilution into complete raw of the 96-well plate). In addition, levooxacin (20 mg per disc) was used as reference drug. The un-inoculated media tested as negative control, aer 24 h incubation at 37 C. MIC was determined by the addition of 40 mL of p-iodonitrotetrazolium violet chloride (INT) (0.2 mg mL À1 , Sigma-Aldrich) to the plates and re-incubated at 37 C for 30 min., the lowest concentration which banned color change is the MIC. 40 In silico docking approach Molecular docking study 41,42 of ligand molecules was carried out to evaluate their binding geometries with the target enzyme avoHB. To understand the binding mode of interactions of newly synthesized compounds with the target, the crystallographic structure of the enzyme was downloaded from the Protein Data Bank (PDB: 3OZU). 38 Additionally, the 2D structures of the ligand molecules were sketched using ChemDraw 16, and converted to SDF format using Open Babel GUI. 43 The docking approach was carried out using PyRx-virtual screening tool. 44 To obtain the 2D and 3D intermolecular representations of the docked compounds, the discovery studio 3.5 was employed. Moreover, in silico ADMET and drug-likeness prediction of the molecules were performed using admetSAR, mol inspiration, and SwissADME free accessible tools.

Conclusion
Novel series of nitrogen-based heterocycles attached to pphenolic unit was synthesized. In vitro study showed that compounds 6a and 6b had appreciable antibacterial activities close to standard drug against all types of bacteria strains tested. Consequently, in silico study revealed that compounds 6a and 6b showed good binding energies against the target enzyme. Overall; the ndings exhibited that compounds 6a and