Synthesis, Characterization, Antibacterial Activity, and Computer-Aided Design of Novel Quinazolin-2,4-dione Derivatives as Potential Inhibitors Against Vibrio cholerae

Cholera is a bacterial disease featured by dehydration and severe diarrhea. It is mainly caused by alimentary infection with Vibrio cholerae. Due to the wide applicability of quinazolin-2,4-dione compounds in medicinal and pharmaceutical chemistry, a new series of N-containing heterocyclic compounds was synthesized. We used the in silico docking method to test the efficacy of quinazolin-2,4-dione compounds in the prevention of cholera in humans. The newly synthesized compounds showed strong interactions and good binding affinity to outer membrane protein OmpU. Moreover, the pharmacokinetic properties of the newly synthesized compounds, such as absorption, distribution, metabolic, excretion, and toxicity (ADMET), were predicted through in silico methods. Compounds with acceptable pharmacokinetic properties were tested as novel ligand molecules. The synthesized compounds were evaluated in vitro for their antibacterial activity properties against Gram-negative Escherichia coli O78 strain using the minimum inhibition concentration (MIC) method. Compounds 2 and 6 showed reproducible, effective antibacterial activity. Hence, our study concludes that the quinazolin-2,4-dione derivatives 1 to 8 may be used as promising drug candidates with potential value for the treatment of cholera disease.


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Evolutionary Bioinformatics study focused on the screening of the newly synthesized compounds against the target protein. PyRx tool is one of such programs which is used to predict the strength of intermolecular docking interactions between the ligand molecules and the binding site of the target protein. 17 In addition, pharmacokinetics properties of the compounds were also in silico predicted.
To extend the applicability of newly quinazolindione derivatives 1 to 8 as antibacterial agents, these newly synthesized compounds were then tested against Escherichia coli O78 strain as a model example for the pathogenic Gram-negative bacteria that are very close to V. cholerae. Our work aims at offering potential inhibitors, which are novel, cheap, and have less side effects, against Homo sapiens OmpU which can be used as drugs for the treatment of cholera disease.

Results and Discussion
Herein, we reported the synthesis of some new quinazolindione derivatives, and in silico molecular docking study was carried out to identify their activity toward cholera disease.

Chemistry
Eight new quinazolin-2,4-dione derivatives were synthesized by (4-methylene-2-oxo-3-phenyl-3,4-dihydro-2H-quinazolin-1-yl)-acetic acid hydrazide 1 as starting material with different reagents. The title compounds 1 to 8 were synthesized according to Schemes 1 and 2. The spectral data (infrared [IR], 1 H-NMR, 13 C-NMR, and mass spectrometry [MS]) of the compounds are represented in Figures S1-S17 (see Supplementary data section). The reaction between N-phenylquinazolin-2,4-dione and ethyl chloroacetate in dimethyl formamide (DMF) containing potassium carbonate furnished 2,4-dioxo-3-phenyl-3,4-dihydroquinazolin-1(2H)-yl acetate, which on treatment with hydrazine hydrate in refluxing EtOH afforded the hydrazide derivative 1. The chemical structure of this hydrazide was established based on its elemental and spectral analyses. The recorded mass at m/z 310.31 corresponding to the formula C 16 H 14 N 4 O 3 , beside Infrared spectrum of hydrazide 1, showed the characteristic absorption bands at wave numbers 3400, 3239, 1687, and 1660 cm −1 to indicate the presence of NH 2 , NH, and C=O's functions, respectively. Furthermore, the strong clue for the structure forthcoming from the 2 singlet signals in the 1 H-NMR spectrum, each integrated for 2 protons and resonated at δ 3.2 and δ 4.3 ppm, clearly indicated the amino and methylene groups. The aromatic protons resonate as multiplet signals at δ 7.2 to 8.0 ppm. The proton of the NH group was verified as a singlet at δ 11.5 ppm. The synthetic strategy pathway for synthesis of hydrazide 1 is reported earlier in previous works. [18][19][20][21] Then, compound 1 was used as starting material for synthesis of new compounds 2 to 8 as declared in Scheme 1. Furthermore, the new heterocyclic moiety pyrazole in compound 2 was prepared via Knorr pyrazole synthesis by treatment of compound 1 with β-di-ketone such as acetylacetone in 79% yield as reported in Scheme 2. 22 The compound 2 was identified from spectral and elemental analyses. Thus, the IR spectrum exhibited the characteristic absorption bands at 1664 and 1652 cm −1 , indicating the presence of functional groups C=Os. The 1 H-NMR of derivative 2 displayed singlet signals at δ 1.3 and δ 4.3 ppm, indicating the presence of 2 methyl and one methylene groups. The aromatic protons resonated as multiplet signals at δ 7.10 to 8.07 ppm. Also, when compound 1 was treated with benzene and/or toluene, sulphonylchloride afforded benzene sulphonyl N′-[2-(4-Methylene-2-oxo-3-phenyl-3,4-dihydro-2H-quinazolin-1yl)-acetyl]-hydrazide 3 and Toluenesulphonyl N′-[2-(4-Methylene-2-oxo-3-phenyl-3,4-dihydro-2H-quinazolin-1-yl)acetyl]-hydrazide 4. Similar spectral analyses were conducted for derivatives 3 and 4. Infrared spectrum exhibited the same characteristic absorption bands at close ranges for the functional groups NH and C=O's groups. The 1 H-NMR spectrum of derivatives 3 and 4 is similar to each other. Extra peak as singlet signals at δ 1.3 ppm indicates the presence of methyl group in compound 4. The starting material 1 reacted with an equimolar amount of ethyl cyanoacetate to yield compound 5. The IR spectrum of the isolated product confirmed the presence of an intense absorption band centered around 2270 cm −1 because of (C≡N) cyano group. Its mass spectrum declared the molecular ion at m/z 377 corresponding to its molecular formula C 19 H 15 N 5 O 4 . Furthermore, the reaction of hydrazide 1 with benzyl chloride was achieved to afford the corresponding (2,4-dioxo-3-phenyl-3,4-dihydro-2H-quinazolin-1-yl)-acetic acid N′-benzylhydrazide 6. Structure proof of compound 6 was deduced from its spectral analysis, where its mass spectrum recorded molecular ion peak (C 23 H 20 N 4 O 3 ) at m/z 400, while the IR spectrum showed characteristic absorption bands at 3415 and 1652 to 1670 cm −1 because of the presence of (NH) and C=Os, respectively. However, treatment of key compound 1 with acetyl chloride and chloroacetyl chloride afforded acetic acid N′-[2-(2,4-dioxo-3-phenyl-3,4-dihydro-2H-quinazolin-1-yl)acetyl]-hydrazide 7 and chloro acetic acid N′-[2-(2,4-dioxo-3phenyl-3,4-dihydro-2H-quinazolin-1-yl)-acetyl]-hydrazide 8. The structural confirmations of these compounds were proved by elemental analysis and spectral data as declared in the "Experimental" section.

In silico study
The outer membrane protein OmpU is selected as a target protein for the identification of novel leads against cholera disease. Recently, the crystal structure of the outer membrane protein OmpU (PDBID: 5ONU) of 341 amino acid residues, with a high resolution, was reported by Li et al, which is a significant virulence factor in V cholerae and delivers a vigorous mechanical basis for the colonization of host cells surfaces and the phagerecognition mechanism. The crystallographic structure of OmpU was downloaded from protein data bank website (rcsb. org) as shown in Figure 1.
Furthermore, the physicochemical properties of the target protein were predicted using the ProtParam tool. 23 The El-Naggar et al 3 molecular weight was 36 645 Da and the isoelectric point (pI) was 4.46. The results show that the amino acid residues ALA, GLY, ASP, ASN, TYR, and VAL were present in high percentages in OmpU protein. In molecular docking method, all the synthesized compounds were docked into the binding site of OmpU. The binding site prediction tools such as MetaPocket and ProBiS declare that the amino acid residues ALA14, ASP29, ARG107-TYR110, ASN125-ASP126, and ARG155-LYS172 are crucial for OmpU to bind with quinazolindione derivatives. An in-house database of 8 quinazolindione compounds was created in standard file format (SDF). All the compounds were energy minimized using Universal Force Field (UFF), 24 to obtain structures with proper bond length between different atoms, and then used in the virtual screening study. In Scheme 1. Synthesis of quinazolindione compounds 2 to 8. (i) Acetylacetone, AcOH, EtOH, and reflux; (ii) benzenesulphonyl chloride, pyridine, and reflux; (iii) toluenesulphonyl chloride, pyridine, and reflux; (iv) ethyl cyanoacetate, EtOH, and reflux; (v) benzyl chloride, EtOH, and reflux; (vi) acetyl chloride, EtOH, and reflux; and (vii) chloroacetyl chloride, EtOH, and reflux.
addition, doxycycline was chosen as a reference compound to compare the docking score of synthesized compounds. Herein, PyRx software was employed to simulate the ligand into the active site of the protein to calculate the binding energy of the ligand-receptor complexes. Computer-based docking predicted 9 conformers for each ligand-protein complex. The preferable binding orientation between protein and compound, with more negative binding affinity score, was considered for further study. 25  El-Naggar et al 5 8 exhibited pi-pi interactions with PHE85. The results showed that all compounds 1 to 8 form pi-cation interactions with the target proteins through ARG107, LYS108, and ARG155. This is due to arginine containing positively charged guanidinium group H 2 N-C(=NH)-NH-that is involved in forming pication interaction with pyrimidine moiety in compounds 1 to 7. However, lysine contains a positively charged amino on its side chain that is involved in forming pi-cation interaction with pyrimidine moiety of compound 8. Clearly, pyrimidine and amide moieties are observed to be common pharmacophore groups, which interact with the binding residues of OmpU protein through various interactions as represented in Table 1. However, doxycycline which is used as a reference has the binding affinity (−10.7 kcal/mol) closer to the synthesized compounds and exhibited 4 H-bond interactions with TYR20, GLY219, and SER239, respectively. It was observed that all the compounds showed strong interactions to OmpU through hydrogen and non-covalent bonds such as pi-interactions. Lower binding energy and more non-covalent bonds imply best binding stability and inhibitory characteristic 26,27 of the synthesized compounds against cholera.
Drug-likeness scores of the screened quinazolindione compounds were filtered by in silico prediction of ADMET, using admetSAR. 28 The compounds with acceptable ADMET properties in the permissible range are considered as novel molecules. 29 The pharmacological properties of 8 compounds 1 to 8 are presented in Table 2. Interestingly, all the newly synthesized compounds have a molecular weight in the range of 310 to 464 (<500). Blood-brain barrier (BBB) + value describes the ability of the compounds to cross the BBB, which is in the permissible ranges for all compounds. Also, the values show that the compounds can be absorbed by the human intestines and noncarcinogenic. The results show that these compounds show better inhibition properties against OmpU protein and promising pharmacokinetics properties.
Our study concluded that the newly synthesized quinazolin-2,4-dione compounds such as (4-methylene-2-oxo-3-phenyl-3,4-dihydro-2H-quinazolin-1-yl)-acetic acid hydrazide 1, hydrazide 8 are of great importance and good inhibitors, which can be used as potential drug-like molecules against cholera. Hence, the OmpU function as pore selectivity helps V cholerae for persistence within diverse extracellular environments, which consider a challenge faced by a respective host (human). The distinct quinazolindione structural features in this study create an informative platform for blocking the function and biophysical properties of OmpU.

Antibacterial activity of quinazolindione compounds against E. coli O78 strain
Eight newly synthesized compounds 1 to 8 were screened for their in vitro antibacterial activities against Gram-negative strain (E. coli O78 strain) by using the minimum inhibitory concentration (MIC) method. The relationship between the reactivity and binding affinity of all synthesized compounds against bacterial specie was evaluated and summarized as MIC values in µg/mL in Figure 3 and Table 3.

The MICs
The colorimetric iodonitrotetrazolium (INT)-formazon assay of compounds 2 and 6 showed reproducible, efficacious antibacterial activity with MICs values of 20 and 10 µg/mL, respectively, against the tested E. coli strain as represented in Figure S18 in the Supplementary data section. The result implicated that the presence of pyrazole moiety and benzyl group is essential for the antibacterial activity of these compounds. 30,31 Methods Chemistry Materials and apparatus. All melting points of the compounds were determined on Griffin apparatus and are uncorrected. The IR spectra of samples were recorded in KBr via a Shimadzu FT-IR 8101 PC infrared spectrophotometer. The NMR spectra were run at 400 MHz using tetramethylsilane (TMS) as the internal standard. Chemical shifts were measured in ppm (δ) related to TMS (0.00 ppm). Mass spectra were measured on a GCMS-QP1000 EX spectrometer at 70 eV. Elemental analyses were carried out at the Micro Analytical Center of Cairo University, Giza, Egypt. Thin-layer chromatography (TLC) was conducted on 0.25 mm precoated silica gel plates (60F-254). Purities of these compounds were established by TLC.

In Silico Study Binding site identification
Various computational methods were used for the prediction of drug-like molecules against cholera. The binding site region was identified using computational prediction tools like MetaPocket2.0 and ProBiS. 32,33 A grid with dimensions 25A° × 25A° × 25A° is created around the binding site of OmpU to perform the screening studies.

In silico docking and ADME prediction
Next, in silico molecular docking of 8 quinazolindione derivatives and reference compound onto the target has been carried out using PyRx-virtual screening tool. The intermolecular interactions between the docked compounds-protein complexes such as H-bonds, π-π, π-cation, and π-sigma interactions add more stability to the target protein. 34 Because of these interactions, compounds are arranged in a certain position within a protein. Discovery Studio ® Visualizer 2016 software (Accelrys Software Inc., San Diego, CA, USA) was used to visualize the interactions. In addition, the prediction of absorption, distribution, metabolic, excretion, and toxicity (ADMET) properties plays a key role in drug design. Bloodbrain barrier, human intestinal absorption (HIA), Caco-2 Permeability (Caco2+), Ames toxicity and carcinogenicity were calculated using admetSAR (http://lmmd.ecust.edu.cn/ admetsar1/). The promising results for the tested compounds indicate that they can be used as drug candidates.

Biological screening (antibacterial activity)
The antibacterial activity of compounds 1 to 8 was estimated by the minimum inhibition concentration (MIC) method. Escherichia coli O78 is the test strain selection pathogenic bacteria in our work. Escherichia coli was kindly donated from the bacteriology Lab.-Faculty of Science, South Valley University. An amount of 0.2 g of each compound was dissolved in 1 mL dimethyl sulfoxide (DMSO) and completed with sterile tryptic soy broth (TSB) medium to 10 mL to avoid the inhibitory effect of DMSO (10-fold dilution). 35 Overnight culture of E. coli was diluted to 1:10.000 into TSB. Bacterial growth was placed into 96-well plates plus different amounts of quinazolindione derivatives. After 24 hours of incubation at 37°C, the MIC was the lowest concentration that inhibited bacterial growth. To confirm bacterial growth inhibition and lack of metabolic activity, 40 µL of p-iodo nitrotetrazolium violet (INT) (0.2 µg/mL; Sigma-Aldrich) was added to the microplate wells and re-incubated at 37°C for 30 minutes. 36 The MIC in the INT assay was defined as the lowest concentration that prevented color change as described earlier by Lall et al. 37

Conclusions
Cholera is common in places with famine and poor sanitation that can cause acute diarrhea. This study provides an insight into the efficiency of quinazolindione derivatives as new chemical entities (NCEs) against cholera. The resulted data from in silico study are helpful to identify new potential lead molecules for the treatment of cholera throughout the blocking mechanisms of OmpU binding sites. The antibacterial activities of the newly synthesized compounds were evaluated and revealed that the compounds 2 and 6 are the most effective against Gram-negative bacterial strains.