Synthesis of Novel pyrimido[4,5-b]quinoline-4-one Derivatives and Assessment as Antimicrobial and Antioxidant Agents

The compounds based on scaffold of quinolines have been reported to possess a wide range of pharmaceutical properties1-7. Several structures based on quinoline have proved effective inhibitors of important proteins from microbial pathogens8. The modified classes of compounds based on quinolines have been studied recently for their antimicrobial9,10. Quinoline-carboxamide I, II, III were reported as the most potent EGFR inhibitors with IC50 2.6, 0.49 and 1.73 mM, respectively 11. Iminosugar/Azasugars fused benzo [1,3]thiazin4-one exhibited significant HIV-RT inhibitory activities12,13 (Figure 1).

Based on that, we decided to complete the work on pyrimido [4,5-b]quinoline and synthesis of benzo[d] [1,3]oxazin-4-one as stating material for new compounds and evaluating their antioxidant and antimicrobial activity.

Equipments
All melting points are uncorrected and were taken on open capillary tubes using electrothermal apparatus 9100. Elemental micro analyses were carried out at microanalytical unit, Central Services Laboratory, National Research Centre, Dokki, Cairo-Egypt, using Vario Elementar and were found within + or -0.5% of the theoretical values. Infrared spectra were recorded on a Jasco FT/IR-6100, Fourier Transform Infrared Spectrometer at cm -1 scale using KBr disc technique at the Central Services Lab. NRC, Dokki, Cairo, Egypt. 1 HNMR spectra were determined by using a JEOL EX-270 NMR Spectrometer at Central Services Lab, NRC. Mass spectra were measured with Finnigan M A T SSQ-7000 mass spectrometer at the Central Services, NRC Dokki, Cairo, Egypt. Follow up of the reactions and checking the purity of the compounds were made by TLC on silica gelprecoated aluminum sheets (Type 60 F254-Merck, Darmstadt, Germany) and the spots were detected by exposure to UV Lamp at 254 nanometer for few seconds.

Synthesis of compounds 9a-d
A solution of benzoxazinone 2 (3.93 g, 0.01 mole) and amine derivatives namely, hydrazine hydrate, p-aminopyridine, 4-bromoaniline, or 4-aminoacetophenone (0.02 mole) in absolute EtOH (30 mL) was refluxed for 6 hours. The solid product that separated on cooling was filtered off, dried and recrystallized from ethanol to afford the quinazolinone derivative 9a-d.

Synthesis of 23a,b
A mixture of quinazolinone 7a (0.01 mole) and maleic anhydride or phthalic anhydride (0.01 mole) was fused in an oil bath at for 6 hrs. The reaction mixture was triturated with ice/HCl. The solid product was filtered off, washed with water several times, dried and then recrystallized from ethanol affording 23a,b.

Test microorganisms
Standard strains used to evaluate antimicrobial activity; Gram positive bacteria; (Bacillus subtilis ATCC 6633 and Staphylococcus aureus ATCC 6538-P), Gram negative bacteria (Pseudomonas aeruginosa ATCC 27853 and Bordetella pertussis ATCC 9797), yeasts (Candida albicans ATCC 10231 and Saccharomyces cervesiae) and fungi (Aspergillus niger NRRL A-326 and Trichoderma viride NRC 314) were obtained from culture collection stocks maintained in the Department of Microbial Chemistry, National Research Centre, Egypt. Bacteria were maintained at 4 °C on nutrient agar slants containing (g/L): beef extract, 3; peptone, 5 and 1 L of distilled water, and adjusted at pH 7.2 before autoclaving (LAC-J0805 autoclave, Daihan Labtech Co., Korea) at 121 °C for 15 min, while yeast and fungi were maintained on Sabouraud dextrose agar (SDA) and Potato dextrose agar (PDA) media, respectively.

Antimicrobial activity
The antimicrobial activity of each chemical compound was investigated in vitro by the Department of Microbial Chemistry, National Research Centre using the agar well diffusion method (WDM) recommended by the Clinical and Laboratories Standards Institute (CLSI) to measure in vitro susceptibility of bacteria to antimicrobial agents used in clinical settings. The accuracy of this test depends on the maintenance of standard procedures. In the present study, a stock solution containing 20 mg/mL in DMSO is prepared for each chemical compound. Dispense nutrient agar seeded with 1.5 × 10 8 CFU/mL of each bacterial strain, SDA seeded with 2.0 × 10 5 CFU/mL of each yeast and PDA seeded with 2.0 × 10 4 CFU/mL for each fungal strain (cooled below 45 ºC) into sterile Petri dishes, give a depth of 4 mm (~20 mL in Petri dish of 85 mm in diameter). Allow the agar to set before moving the plates. Agar wells of diameter 8 mm were made in the agar plates with the help of a sterilized cork borer. Wells were loaded with 100 µL (20 mg/mL) of tested compound solutions and controls under aseptic condition. These plates were sealed with parafilm and kept in the refrigerator for 4 h at 5 °C for the complete diffusion of antimicrobial compounds, if any.
Thereafter, the sealed plates were incubated upright at 35 °C for 18-24 h for bacteria and yeasts, and 48-72 h at 28 °C for fungi. Positive control experiments were conducted under similar conditions using cefaxone (20 mg/mL), Ketoconazole (20 mg/mL) and cyclosporine (10 mg/mL) as standard drugs for antibacterial and antifungal activity, respectively. Similarly, 10 μL DMSO was used as a negative control. After the incubation period, antimicrobial activity was evaluated by measuring the diameter of inhibition zone in millimeters (mm) and compared to that of the standard (Positive controls). Inhibition zones with a diameter ≥ 16 mm were considered to have antimicrobial activity for further quantitative tests of their activity. The experiment was performed in triplicate and the average inhibition zone was calculated.

Determination of minimal inhibitory concentration (MIC)
In microbiology, the minimum inhibitory concentration (MIC) endpoints were defined as the lowest concentration of the assayed antimicrobial agent, which resulted in a 100% reduction in growth compared to the antimicrobial agent-free growth control test 23 . The bacteriostatic activity of the active chemical compounds (with inhibition zones ≥ 16 mm) was evaluated using a two-fold serial dilution technique 24 . Two-fold serial dilutions of the tested compound solutions were prepared using the proper nutrient broth. The final concentrations of the solutions were 25, 50, 75, 100, 150, 200 and 300μg/mL. Each 5.0 mL received 0.1 mL of inoculums and incubated at 37 °C for 24 h for bacteria and yeasts, and 48 h at 28 °C for fungi. Tests were performed in triplicate and repeated twice. The lowest concentration showing no growth was considered the minimum inhibitory concentration (MIC) ( Table 3).

Evaluation of antioxidant activity using the DPPH radical scavenging method
The percentage of antioxidant activity of each chemical compound was measured by the Department of Microbial Chemistry, National Research Centre using the 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay 25 . This assay is based on the measurement of the ability of antioxidants to reduce DPPH by measuring the decrease in its absorption 26 . DPPH reacts with hydrogen/electron donor compounds and has a maximum UV-Vis absorption of 515-520 nm 27 . The reaction mixture consisted of 50 µL (10-200 µg/mL) of each chemical compound dissolved in dimethyl sulfoxide (DMSO), as well as the reference standard ascorbic acid and the volume was made uniformly to 150 µL using ethanol, 3 mL of absolute ethanol and 150 µL of freshly prepared DPPH radical solution (0.5 mM in ethanol). The mixtures were shaken vigorously and left to stand in the dark for 30 min at room temperature, and the absorbance was measured at 517 nm in Cary-100 UV-Vis spectrophotometer (Agilent Technologies, Frankfurt, Germany) using ethanol as a blank. Control reactions were performed without the test sample (i.e. 150 µL of DPPH + 3.0 mL ethanol). The experiment was carried out in triplicate for each chemical compound. Radical scavenging capacity was expressed as a percentage (%) and was calculated using the following formula: Radical scavenging activity (%) = Abs control and Abs sample : the absorbance values of the control as well as the sample.
\The antioxidant activity of each chemical compound and ascorbic acid was expressed as EC 50 (the effective micromolar concentration required to scavenge 50% of DPPH radicals) is a typically employed parameter to express the antioxidant capacity and to compare the activity of different compounds 28 (Table 1). It is worth note that EDTA was added to prevent ascorbic acid oxidation.

Chemistry
Compound 1 was synthesized previously by the author 29 . In scheme 1, anthranilic acid (2) reacted with excess of acid chloride derivatives (1) in presence of dry pyridine to afford quinolin-oxazin-4-one derivative 30 (3). IR spectrum of compound 3 demonstrated two bands of C=O and NH at ≈ 1715, 1745 and 3450 cm -1 , respectively (Scheme 1). 1 H-NMR of 3 showed singlet tow OCH 3 and NH signals occurring at 3.72, 3.80 and 10.5 ppm, respectively. Compound 3 play as electrophilic intermediate key for the synthesis of interest pharmaceutical derivatives.
In scheme 4, The nucleophilic amino group of compound 9a condensed with series of aldehydes (16a-d) in presence of piperidine to afford benzylidene-quinazoline (17a-d). Also, 9a refluxed in dry pyridine with CH 3 COCl or phCOCl to afford 19, 21a, b, respectively (Scheme 4). In addition to, 9a fused with succinic anhydride and phthalic anhydride (22a, b) to yield the corresponding compounds 23a, b.
In last scheme 5, hexahydropyrimido [4,5-b]quinolin was prepared by refluxing of compound 17a, b with CH 2 COCl 2 in EtOH in presence of Et 3 N to afford compounds 25a, b. In addition to, compound 17a, b refluxed with thioglycolic acid or thiosalicylic acid in dry benzene to yield new thiazolidin-4-one 27a, b and thiazin-one 29a, b, respectively.