Enantioselective synthesis, characterization, molecular docking simulation and ADMET profiling of α-alkylated carbonyl compounds as antimicrobial agents

All living organisms produce only one enantiomer, so we found that all natural compounds are presented in enantiomerically pure form. Asymmetric synthesis is highly spread in medicinal chemistry because enantiomerically pure drugs are highly applicable. This study initially demonstrated the feasibility of a good idea for the asymmetric synthesis of α-alkylated carbonyl compounds with high enantiomeric purity ranging from 91 to 94% using different quinazolinone derivatives. The structure of all compounds was confirmed via elemental analysis and different spectroscopic data and the enantioselectivity was determined via HPLC using silica gel column. The synthesized compounds’ mode of action was investigated using molecular docking against the outer membrane protein A (OMPA) and exo-1,3-beta-glucanase, with interpreting their pharmacokinetics aspects. The results of the antimicrobial effectiveness of these compounds revealed that compound 6a has a broad biocidal activity and this in-vitro study was in line with the in-silico results. Overall, the formulated compound 6a can be employed as antimicrobial agent without any toxicity with high bioavailability in medical applications.


Synthesis of 3-ethylhexan-2-one (6a, b)
A mixture of 5a, b (2.1 g, 5.0 mmol) was dissolved in Dioxane (20.0 mL), then methane sulphonic acid (few drops) was added and refluxed for 12 h, the product precipitated by adding ice, filtrated off, then compounds 6a, b were extracted from the filtrate via methylene chloride.

General procedure for the synthesis of quinazolinone esters (8a, b)
In 100 mL round bottom flask a mixture of compound 3 (2.6 g, 10.0 mmol), carboxylic acid derivatives (10.0 mmol), methane sulphonic acid (few drops) and 50.0 mL benzene was refluxed for 4 h using Dean-Stark trap (TLC control), then the reaction mixture was basified with sodium carbonate, filtered, and dried 4 .

General procedure for the synthesis of 2-ethylpentanoic acid (10a, b)
A mixture of compound 9a, b (0.6 g, 5.0 mmol) and methane sulphonic acid (few drops) in 20 .0mL Dioxane was refluxed for 12 hours.Finally, the reaction mixture was poured into ice water, filtered and the target product was extracted with methylene chloride, dried with anhydrous sodium sulphate to give pale yellow liquid 14

General procedure for the synthesis of 2-ethyl-N-((R)-2-hydroxy-1-phenylethyl) pentanamide (11a, b)
Compound 10a, b (0.6 g, 5.0 mmol) was added to (2.2 mL, 30.0 mmol) of thionyl chloride.The reaction mixture was refluxed for 4 hours and the excess of thionyl chloride was evaporated, and the acid chloride was dissolved in 10.0 mL THF. then the acid chloride was added drop wise to a solution of optically active (R)-phenyl glycinol (0.7 g, 5.0 mmol) in 10.0 mL THF, then the reaction mixture was refluxed for 6 h (TLC control), the diastereomeric amide (11a, b) was extracted with methylene chloride, washed with 1 N HCl then 1N NaOH, filtered and dried over sodium sulphate anhydrous 14 .

Antimicrobial activities
The evaluated bacterial and fungal strains were tested for susceptibility to the synthesized compounds by measuring the diameter of the inhibitory zone using the agar well diffusion technique.The compounds were synthesized and then dissolved in solutions containing 0.1% DMSO at a concentration of 60 mg/mL.To compare the effectiveness of the bacterial and fungal strains, respectively, Ampicillin (5 µg) and Clotrimazole (10 µg) were used as positive controls, and 0.1% DMSO was used as a negative control.The bacterial and fungal strains were sub-cultured in nutrient broth medium for an entire day before being concentrated to 10 6 CFU/mL at 630 nm.A 100 µL aliquot of each broth culture was evenly seeded throughout the nutrient agar medium using a sterile disposable plastic rod.On the surface of the nutritional agar medium, 9 mm wells were successfully made using a sterile cork porer, and 50 µL of each compound was then added 17 .The % activity index for the complex was calculated by the formula as follow: www.nature.com/scientificreports/

Statistical analysis
Data have been analyzed using the GraphPad Prism software (San Diego, CA, USA) (GraphPad Prism 6, https:// www.graph pad.com/ scientific-software/ prism/).The experimental data are expressed as mean ± SE; n = 3.The significance of the difference was analyzed using the one-way ANOVA.The acceptable significance was recorded when the p-value was < 0.05.

Chemistry
The general approach for the synthesis of the designed compounds was illustrated in Figs.  3) respectively.The chemical structure of compounds 2 and 3 was established according to their elemental analysis and spectral data as described previously by Vijayakumar 12 and Yang 13 respectively.As described in Fig. 4, the chemical modification of compound 2 with 2-pentanone or 2-hexanone in acidic medium led to formation of Z-3-(4-hexan-2-ylideneamino) phenyl)-2-phenylquinazolin-4-(3H)-one (4a) and Z-3-(4-pentan-2-ylideneamino) phenyl)-2-phenylquinazolin-4-(3H)-one (4b) respectively whose structures were determined on the basis of their elemental analysis and spectral data.The FT-IR spectrum of compound 4a revealed absorption band at 1534 cm −1 characteristic for C=N group, 1642 cm −1 identical to C=O group, 2923 cm −1 due to CH-Aliphatic, 3090 cm −1 due to CH-Aromatic and a complete loss of NH 2 stretching.The 1 H NMR spectrum exhibited triplet signal at δ 0.80 ppm characteristic of methyl protons (CH 3 ) , multiplet signals at δ 1.15-1.30ppm characteristic of methylene protons (2CH 2 ), triplet signal at δ 1.40 ppm characteristic of methylene protons (CH 2 ) and singlet signal at δ 2.01 ppm characteristic of methyl proton (CH 3 ).The 13 C NMR spectrum of compound 4a showed signals at δ 170.65 and δ 165.19 which can be assigned to 2C=N groups.The FT-IR spectrum of compound 4b showed absorption band at 1587 cm −1 due to C=N group, 1682 cm −1 for C=O group and a complete loss of NH 2 stretching.The 1 H NMR spectrum exhibited triplet signal at δ 0.80 ppm characteristic of methyl protons (CH 3 ) , multiplet signals at δ 1.12 ppm characteristic of methylene protons (CH 2 ) , triplet signal at δ 1.29 ppm characteristic of methylene protons (CH 2 ) and singlet signal at δ 2.02 ppm characteristic of methyl proton (CH 3 ).The 13 C NMR spectrum exhibited signals at δ 170.70 and δ 165.19 which can be assigned to 2C=N groups.
As illustrated in Fig. 11, The chemical reaction of compounds 10a, b with thionyl chloride led to formation of 2-ethylpentanoyl chloride which on reaction with optically active (R)-phenyl glycinol led to formation of 2-ethyl-N-((R)-2-hydroxy-1-phenylethyl) pentanamide (11a, b).The structure of these compounds was established according to their elemental analysis and spectral data.
Furthermore, compounds 7a, b and 11 a, b were separated via HPLC using silica gel column and give high values of enantiomeric ratio indicating that compounds 2, 3 enhanced the steric hindrance during asymmetric alkylation process as tabulated in Table 1.www.nature.com/scientificreports/

In-silico and ADMET pharmacokinetics studies
The newly synthesized ligand molecules and the target proteins were docked together to learn more about the chemical interactions and binding scores 18 .Here, exo-1,3-beta-glucanase and outer membrane protein A (OMPA) are recognized as interesting therapeutic targets for the development of antibacterial and antifungal drugs.To understand the ligand compounds' mode of action as antibacterial agents in the current work, the docking method utilized OMPA protein.The findings revealed that compound 6a had the best binding affinity against the target OMPA protein (ΔG = −8.23 kcal mol −1 ) and the most promising bacterial inhibitory effect among all other newly synthesized compounds compared with the reference ampicillin FDA approved antibacterial drug that gave binding energy equal to −10.05 kcal mol −1 .Compound 6a bonded with the OMPA protein residues PHE353, LYS361, LEU352, ARG447, ARG405, ARG448, ILE351, GLU444 via hydrogen bonding and electrostatic interactions compared with the ampicillin reference drug that bonded to the OMPA target protein residues GLU444, PHE353, GLY356, ARG405, ARG447, THR355, LEU401, LEU352 by hydrogen bonding and electrostatic interactions.Moreover, to investigate the mechanism of action of the synthesized compounds as antifungal medicines, exo-1,3-beta-glucanase target protein was used in the docking simulation.The findings revealed that compound 6a had the strongest binding affinity for the target exo-1,3-beta-glucanase protein (ΔG = −9.25 kcal mol −1 ) and the most promoting inhibitory activity of fungi compared with the clotrimazole reference FDA antifungal drug that gave binding energy equal to −9.89 kcal mol −1 .Compound 6a bonded to exo-1,3-beta-glucanase with hydrogen bonding, π and electrostatic interactions to the residues TRP363, TYR255, PHE144, ASN146, HIS135, GLU192, TRP373 compared with reference clotrimazole drug that bonded also with hydrogen bonding, π, and electrostatic interactions to the exo-1,3-beta-glucanase target protein residues ASP145, PHE258, PHE144, ASN146, ASN305.Thus, compound 6a elucidated dual antibacterial and antifungal potent effects, according to the results elucidated in Table 2. Figures 12 and 13 showed the 2D and 3D molecular interactions network of newly synthesized compounds and reference drugs with the target proteins.
Furthermore, all synthetic compounds need to have their ADMET-determined pharmacokinetic characteristics.A radar map with 13 parameters was used to assess each compound's bioavailability and physicochemical properties (Fig. 14).All the investigated compounds met all the criteria for good permeability, the Ro5 (no. of violations 0), and adequate oral bioavailability, as the TPSA range was 17.07-37.300A 2 .They also demonstrated rotatable bonds with numbers ranging from 0 to 10, indicating flexibility.Because their HBA and HBD values were within the acceptable range, they had better solubility in cellular membranes.Log p values < 5 indicated good lipophilicity characteristics.Furthermore, according to the ADMET criterion, the compounds had higher Human Intestinal Absorption (% HIA) ratings, implying that the human gut could absorb them more effectively.Because the target chemicals do not cross the blood-brain barrier, they offer an excellent CNS safety profile.Finally, all AMES toxicity and carcinogenicity test results came back negative, demonstrating their safety (Table 3).Moreover, the in-vitro studies confirmed the AMES toxicity results and elucidated that the IC 50 of all synthesized compounds (6a-10b) on WI-38 (normal lung cell line) was equal to 157.3 ± 1.23, 193.6 ± 2.1, 86.90 ± 2.2 and 117.3 ± 2.3 µM respectively.Also, all compounds were investigated their toxicity on WISH normal cell line and the IC 50 values were equal to 180.5 ± 1.4, 156.0 ± 1.6, 160.2 ± 2.6 and 127.4 ± 2.7 µM respectively as shown in Figs.S13 and S14.Therefore, the insilico and invitro studies were inline with each other and revealing the compounds' safety.

In-vitro antimicrobial activity
Antimicrobial resistance is influenced by outer membrane protein A (OMPA) and exo1,3 beta-glucanase.One of the main α-barrel porins expressed in bacterial outer membranes is called OMPA.This membrane has a variety of roles in the pathophysiology of bacteria, including resistance, induction of host cell death, and adhesion to host cells.Clinically, overexpression of the OMPA gene is linked to the onset of pneumonia and bacteremia, as well as patient death.A globular protein facing the periplasmic space, the C-terminal OmpA-like domain of outer membrane proteins interacts non-covalently with peptidoglycan to maintain cellular integrity and stability 19 .Furthermore, as the primary skeletal polysaccharides of fungal cell walls, β-1,3-glucanase catalyzes the hydrolytic cleavage of the β-1,3-D-glycosidic linkages in β-1,3-glucans.Therefore, inhibiting the OMPA and β-1,3-glucanases during the pathogenicity may appeared to be the primary role for treatment 20 .
According to the previous point of view, there was an urgent need to discover newly synthesized compounds that were effective against these target proteins without any adverse effects.Therefore, our finding elucidated the antimicrobial impact of the newly synthesized carbonyl compounds in-silico against OMPA and exo-β-1,3-glucanases and observed their safety pharmacokinetics profile using AMES examinations.Herein, the invitro disc diffusion antimicrobial method confirmed all the in-silico results as compound (S) 3-ethylhexan-2-one (6a) showed a highly bactericidal effect against all bacterial strains investigated E. coli, S. aureus and B. subtilis with activity index equal to 73±2.5, 85.71±2.72 and 69.56±1.8%respectively compared with ampicillin    and 44±2.7%)respectively compared with ampicillin and clotrimazole reference drugs (Table 4; Fig. 15).Ultimately, we would like to praise that this work was considered a new and modern breakthrough in the science of stereochemistry especially in the discovery of new drugs for antimicrobial treatment.

Antimicrobial structure activity relationship (SAR)
As described in Fig. 16, the structure activity relationship demonstrated that the inhibitory effect on Grampositive, gram negative bacterial and fungal species was due to that the compound could easily cross the peptidoglycan structure of the bacterial and fungal species and then destroy the cell wall structure of them, which leads to the leakage of intracellular substances and invading the bacterial and fungal cells play a bacteriostatic role.Additionally, Increasing the chain of long aliphatic will cause an increase in the corresponding activity 21,22 .• Some chemical reactions proceeded at − 96 °C which need specific cooling device to reached to this tem- perature (we can use a mixture of methanol with liquid nitrogen as a cooling bath if the cooling device is not available) • Most of reactions proceeded under nitrogen gas so, if the nitrogen system is not available, we can use nitrogen ballon at the top of the condenser.• Cleaning, disinfection, and Sterilization for the removal of foreign material from objects is very important before testing the antimicrobial activity because inorganic and organic materials that remain on the surfaces of instruments interfere with the effectiveness of these processes.

Recommendations
Our prospective recommendation is to complete studying these newly synthesized compounds on antibiotic microbial resistance.Also, we strongly recommend investigating their antitumor impact invitro and in vivo in further studies.

Figure 12 .
Figure 12.Molecular docking interactions of all synthesized compounds and reference drug with OMPA protein, 3D-(Left side) and 2D (Right side).

Figure 15 .
Figure 15.Activity index of all synthesized compounds against panel of bacterial and fungal strains.The experiment was done in triplicates and **p < 0.01, ***p < 0.001 and ****p < 0.0001 was significant.

Figure 16 .
Figure 16.Structure activity relationship of the synthesized compounds.

Table 1 .
Enantiomeric values of compounds 7a, b and 11 a, b Reaction conditions: a deprotonation temperature during asymmetric alkylation.bYield of isolated products.cconfiguration of the isolated products.The enantiomeric ratio (e.r.) was determined via HPLC analysis on silica gel column. d

Table 2 .
Calculated docking scores (kcal/mol) of all synthesized compounds with the target proteins.