Discovery of Quinazoline-2,4(1H,3H)-Dione Derivatives as Potential Antibacterial Agent: Design, Synthesis, and Their Antibacterial Activity

In this paper, we report on the design and synthesis of a novel series of quinazoline-2,4(1H,3H)-dione derivatives as fluoroquinolone-like inhibitors of bacterial gyrase and DNA topoisomerase IV to identify and develop antimicrobial agents to prevent bacterial resistance problems. Their structures were confirmed using spectroscopic analyses (IR, NMR, and EI-MS). The novel quinazoline-2,4(1H,3H)-dione derivatives were evaluated for their antimicrobial activities against Gram-positive and Gram-negative bacterial strains using the Agar well diffusion method to study the antimicrobial activities and compared them with the standard drugs. Most compounds displayed moderate activity. Among the tested compounds, the most promising compounds 13 and 15 provided broad bioactive spectrum against Gram-positive and Gram-negative strains compared to the standard drugs.

Fluoroquinolones, for example, I and II, are fatal and decisive antibacterial agents to exceedingly control many bacterial diseases [43]. However, many bacterial strains are resistant to such fluoroquinolone-like compounds [44][45][46]. Therefore, one way to avoid the resistance problems is to identify new derivatives to address the resistant mutant. On the other hand, a comprehensive study proved that quinazoline-2,4(1H,3H)-dione derivatives are fluoroquinolone-like inhibitors of bacterial gyrase and DNA topoisomerase IV [5,47]. In a continuation of our previous work on the synthesis and structural-activity relationships (SAR) of a novel series of 1,3-disubistituted quinoline derivatives [48] due to the current need to identify and develop inhibitors to prevent the bacterial resistance. Therefore, we selected the quinazoline-2,4(1H,3H)-dione scaffold to study and check the substituents' effect at the same 1-and 3-positions on the quinazoline-dione ring (Figure 1). The most active compound discovered in our previous work was compound III, as shown in Figure 1. other hand, a comprehensive study proved that quinazoline-2,4(1H,3H)-dione derivatives are fluoroquinolone-like inhibitors of bacterial gyrase and DNA topoisomerase IV [5,47]. In a continuation of our previous work on the synthesis and structural-activity relationships (SAR) of a novel series of 1,3-disubistituted quinoline derivatives [48] due to the current need to identify and develop inhibitors to prevent the bacterial resistance. Therefore, we selected the quinazoline-2,4(1H,3H)-dione scaffold to study and check the substituents' effect at the same 1-and 3-positions on the quinazoline-dione ring ( Figure 1). The most active compound discovered in our previous work was compound III, as shown in Figure 1.

Chemistry
The structural-activity relationship study of the previous 1,3-disubistituted quinoline derivatives suggested that the antibacterial activity could be increased by performing synthetic modification at the 1-and 3-positions, interestingly, the incorporation of the oxadiazole rings at both positions led to a significantly more potent compound (compd. III, Figure 1). Therefore, one part of this study aimed to investigate the incorporation of the same heterocyclic ring at the 1-and 3-positions of the quinazoline-2,4(1H,3H)-dione scaffold, as depicted in Scheme 1. Appropriately, quinazoline-2,4(1H,3H)-dione (1) was either commercially available or synthesized from anthranilic acid [49], followed by Nalkylation with ethyl chloroacetate in stirring dimethyl formamide in the presence of potassium carbonate at room temperature. The key intermediate 2,2 -(2,4-dioxoquinazoline-1,3(2H,4H)-diyl)di(acetohydrazide) (3) was afforded via the hydrazinolysis reaction of 2 at the reflux condition. Alternatively, synthesized from 2,2 -(2,4-dioxoquinazoline-1,3(2H,4H)diyl)diacetonitrile (4), the hydrolysis of the respective nitrile groups to afford the acid derivative 5, was followed by esterification in the presence of sulfuric acid and methanol. The resulting dimethyl 2,2 -(2,4-dioxoquinazoline-1,3(2H,4H)-diyl)diacetate (6) underwent the hydrazinolysis reaction to afford the key intermediate 3 in a good yield of 72%. The crucial derivative 7 was prepared from dihydrazide 3 by treatment with carbon disulfide in the presence of potassium hydroxide. Finally, the target compounds 8a and 8b were synthesized via the alkylation reaction of compound 7 with benzyl chloride or methyl iodide, respectively. The structure of the synthesized derivatives was confirmed using mass spectrometry and NMR. The structure of the key intermediate 3 was obviously confirmed by the 1 H-NMR spectrum, which showed the demise of the triplet signals at 1.21 ppm as well as the multiple signal at 4.17 ppm arising from the two COOEt groups of compound 2. Moreover, the appearance of signals assignable to the new (CONHNH 2 ) groups at 9. 30, 9.21, and 4.27 ppm. The cyclization of compound 3 resulted into compound 7 which was confirmed by the absence of the last-mentioned signals for (NH & NH 2 ) groups in the 1 H-NMR spectrum. Compounds 8a and 8b were confirmed by the appearance of new signals at 4.44 ppm arising from the new methylene fragments in the 1 H-NMR spectrum for 8a and in parallel with the appearance of a new signal at 2.67 ppm arising from the new methyl fragments for 8b. The key intermediate 3 could provide the opportunity for further novel quinazoline-2,4(1H,3H)-dione derivatives and have been prepared based on the synthetic producers displayed in Scheme 2. The reaction of compound 3 with diethyl ethoxymethylenemalonate or ethyl ethoxymethylene cyanoacetate under the reflux condition in acetic acid affording the malonate derivative 9a or cyanoacetate derivative 9b as a mixture of E/Z isomers, respectively. Furthermore, the direct condensation of diacetohydrazide 3 with aromatic aldehyde benzaldehyde, 2-hydroxybenzaldehyde, or 4-methoxybenzaldehyde to afford the corresponding arylidene 10a, 10b, or 10c, respectively, as mixtures of E/Z isomers. Comparably, the condensation reaction with cyclic ketone cyclopentanone, cyclohexanone, or cycloheptanone afforded the corresponding cycloalkylidene derivatives 11a, 11b, or 11c, respectively. The structure of the novel products 9a and 9b were confirmed by the demise of the two -NH2 groups in compound 3 and the appearance of new signals corresponding to the new methine protons (HN-CH=) at 7.69 and 8. 20-8.21 ppm, respectively, in the 1 H-NMR spectrum, in addition to the appearance of the characteristic signals of the new COOEt groups. Compounds 10a, 10b, and 10c were confirmed by the 1 H-NMR spectrum, in which the signals matching to the two amino groups in compound 3 were The key intermediate 3 could provide the opportunity for further novel quinazoline-2,4(1H,3H)-dione derivatives and have been prepared based on the synthetic producers displayed in Scheme 2. The reaction of compound 3 with diethyl ethoxymethylenemalonate or ethyl ethoxymethylene cyanoacetate under the reflux condition in acetic acid affording the malonate derivative 9a or cyanoacetate derivative 9b as a mixture of E/Z isomers, respectively. Furthermore, the direct condensation of diacetohydrazide 3 with aromatic aldehyde benzaldehyde, 2-hydroxybenzaldehyde, or 4-methoxybenzaldehyde to afford the corresponding arylidene 10a, 10b, or 10c, respectively, as mixtures of E/Z isomers. Comparably, the condensation reaction with cyclic ketone cyclopentanone, cyclohexanone, or cycloheptanone afforded the corresponding cycloalkylidene derivatives 11a, 11b, or 11c, respectively. The structure of the novel products 9a and 9b were confirmed by the demise of the two -NH 2 groups in compound 3 and the appearance of new signals corresponding to the new methine protons (HN-CH=) at 7.69 and 8. 20-8.21 ppm, respectively, in the 1 H-NMR spectrum, in addition to the appearance of the characteristic signals of the new COOEt groups. Compounds 10a, 10b, and 10c were confirmed by the 1 H-NMR spectrum, in which the signals matching to the two amino groups in compound 3 were replaced by singlet signals assignable to the new Schiff base protons (N=CH-Ar) in the region of 8. 12-8.44 ppm, in addition to the new aromatic protons because of the insertion of the aryl group of the appropriate aldehydes in the range of 6.87-8.36 ppm. Compounds 11a, 11b, and 11c showed signals attributable to the new aliphatic protons as a result of replaced by singlet signals assignable to the new Schiff base protons (N=CH-Ar) in the region of 8. 12-8.44 ppm, in addition to the new aromatic protons because of the insertion of the aryl group of the appropriate aldehydes in the range of 6.87-8.36 ppm. Compounds 11a, 11b, and 11c showed signals attributable to the new aliphatic protons as a result of the installation of cyclic aliphatic moieties of the appropriate cyclic ketones at the range 1. .68 ppm.
Scheme 2. The synthesis of the target compounds 9-11.
The reaction of diacetohydrazide 3 with phenyl isocyanate or phenyl isothiocyanate in ethanol under the reflux condition affords the corresponding disemicarbazide derivative 12a or dithiosemicarbazide derivative 12b in a good yield. Consequently, such derivatives could be practical as a template for further modifications to synthesize more substituted analogues at the 1-and 3-positions of the quinazoline-2,4(1H,3H)-dione scaffold, particularly with hetero cyclic moieties. The triazole, oxadiazole, or thiadiazole derivatives can be afforded during the cyclization reaction of 12b based on the reaction medium. The cyclization of the last product 12b in the presence of NaOH under reflux conditions afforded 1,3-bis((5-mercapto-4-phenyl-4H-1,2,4-triazol-3-yl)methyl)quinazoline-2,4(1H,3H)-dione (13). Otherwise, the cyclization of the dithiosemicarbazide derivative 12b in the presence of mercuric oxide HgO under reflux conditions gave 1,3-bis((5-(phenylamino)-1,3,4-oxadiazol-2-yl)methyl)quinazoline-2,4(1H,3H)-dione 14a while the cyclization via stirring at 0 °C in the presence of sulfuric acid provided 1, 3-bis On the other hand, disemicarbazide derivative 12a was easily intermolecular-cyclized, either with 2chloroacetonitrile or ethyl bromoacetate in the presence of anhydrous sodium acetate in absolute ethanol, and provided compounds 15 and 16, respectively, in a very good yield (Scheme 3). Compounds 12a and 12b were confirmed by 1 H-NMR, in which the signals corresponding to the (NH2) protons in compound 3 were replaced by signals assignable to the new (CONHPh or CSNHPh) protons at the range of 9. .00 ppm and the new aromatic protons at the range of 7. 19-8.10 ppm. The cyclization of compound 12b afforded compound 13, which was confirmed by the absence of signal (CSNHPh) protons with the appearance of new typical signals of the (SH) proton at 13.79 ppm. Compounds 14a and 14b were confirmed by the absence of singlet signals at the range of 9. .00 ppm, which was related to the (NHNHCSNH) protons and by the presence of new singlet signals at Scheme 2. The synthesis of the target compounds 9-11.
The reaction of diacetohydrazide 3 with phenyl isocyanate or phenyl isothiocyanate in ethanol under the reflux condition affords the corresponding disemicarbazide derivative 12a or dithiosemicarbazide derivative 12b in a good yield. Consequently, such derivatives could be practical as a template for further modifications to synthesize more substituted analogues at the 1-and 3-positions of the quinazoline-2,4(1H,3H)-dione scaffold, particularly with hetero cyclic moieties. The triazole, oxadiazole, or thiadiazole derivatives can be afforded during the cyclization reaction of 12b based on the reaction medium. The cyclization of the last product 12b in the presence of NaOH under reflux conditions afforded 1,3-bis((5-mercapto-4-phenyl-4H-1,2,4-triazol-3-yl)methyl)quinazoline-2,4(1H,3H)dione (13). Otherwise, the cyclization of the dithiosemicarbazide derivative 12b in the presence of mercuric oxide HgO under reflux conditions gave 1,3-bis((5-(phenylamino)-1,3,4-oxadiazol-2-yl)methyl)quinazoline-2,4(1H,3H)-dione 14a while the cyclization via stirring at 0 • C in the presence of sulfuric acid provided 1, 3-bis On the other hand, disemicarbazide derivative 12a was easily intermolecular-cyclized, either with 2-chloroacetonitrile or ethyl bromoacetate in the presence of anhydrous sodium acetate in absolute ethanol, and provided compounds 15 and 16, respectively, in a very good yield (Scheme 3). Compounds 12a and 12b were confirmed by 1 H-NMR, in which the signals corresponding to the (NH 2 ) protons in compound 3 were replaced by signals assignable to the new (CONHPh or CSNHPh) protons at the range of 9. .00 ppm and the new aromatic protons at the range of 7. 19-8.10 ppm. The cyclization of compound 12b afforded compound 13, which was confirmed by the absence of signal (CSNHPh) protons with the appearance of new typical signals of the (SH) proton at 13.79 ppm. Compounds 14a and 14b were confirmed by the absence of singlet signals at the range of 9. .00 ppm, which was related to the (NHNHCSNH) protons and by the presence of new singlet signals at the range of 10. .53 ppm related to (NHPh) in the 1 H-NMR spectrum. Similarly, compounds 15 and 16 were confirmed by the absence of singlet signals at the range of 8. .09 ppm, which was related to the (NHNHCONH) protons and by the presence of new signal at 4.25 ppm, which was related to the new (NH 2 ) group for compound 15, and new signals at 4.52 and 4.72 ppm, which were related to the new (-OCH 2 CO) groups for compound 16.
the range of 10. .53 ppm related to (NHPh) in the 1 H-NMR spectrum. Similarly, compounds 15 and 16 were confirmed by the absence of singlet signals at the range of 8. .09 ppm, which was related to the (NHNHCONH) protons and by the presence of new signal at 4.25 ppm, which was related to the new (NH2) group for compound 15, and new signals at 4.52 and 4.72 ppm, which were related to the new (-OCH2CO) groups for compound 16. Scheme 3. The synthesis of the target compounds 12-16.

Antibacterial Activity
The antimicrobial activities of the synthesized compounds were examined against some targeted pathogenic microorganisms obtained from the American Type of Culture Collection (ATCC; Rockville, MD, USA). The compounds were evaluated for antimicrobial activity against Staphylococcus aurous , Listeria monocytogenes ATCC-35152 (List.), Escherichia coli ATCC-25922 (E.C.), Salmonella typhi ATCC-15566 (Salm.), and Candida albicans ATCC-10231 (C. Alb.) [50,51]. The stock cultures of pathogens used in this study were maintained on nutrient agar slants at 4°. The Agar well diffusion method was employed to study the antimicrobial activities according to the method described by Kheiralla et al. [52] and Abdelhameed et al. [53] in which the zone of inhibition for the tested compound was measured by a ruler to determine its size (in mm) and compared with that produced by the reference antibacterial drugs ampicillin and vancomycin. The minimum inhibitory concentration (MIC) calculation of the synthesized compounds was

Antibacterial Activity
The antimicrobial activities of the synthesized compounds were examined against some targeted pathogenic microorganisms obtained from the American Type of Culture Collection (ATCC; Rockville, MD, USA). The compounds were evaluated for antimicrobial activity against Staphylococcus aurous ATCC-47077 (St.), Listeria monocytogenes ATCC-35152 (List.), Escherichia coli ATCC-25922 (E.C.), Salmonella typhi ATCC-15566 (Salm.), and Candida albicans ATCC-10231 (C. Alb.) [50,51]. The stock cultures of pathogens used in this study were maintained on nutrient agar slants at 4 • . The Agar well diffusion method was employed to study the antimicrobial activities according to the method described by Kheiralla et al. [52] and Abdelhameed et al. [53] in which the zone of inhibition for the tested compound was measured by a ruler to determine its size (in mm) and compared with that produced by the reference antibacterial drugs ampicillin and vancomycin. The minimum inhibitory concentration (MIC) calculation of the synthesized compounds was also performed according to a slightly modified previous method [54,55] in which the MIC was defined as the concentration at which the bacteria and yeast did not show visible growth with respect to the positive control.
The synthesized intermediate 3 and compound 9b both showed no activity against most of the tested Gram-positive and Gram-negative strains, except for the Candida albicans strain, which is quite sensitive to those derivatives with an inhibition zone value of 11 mm and MIC value of 80 mg/mL, and so more effective than the reference antibacterial drug ampicillin. Compounds 13 and 15 gave broad bioactive spectrum against Gram-positive and Gram-negative strains in which compound 13 with triazole moieties at the 1-and 3-poisitions of the quinazoline-2,4-dione backbone showed moderate activity with a measured inhibition zone value of 9 mm against the Staphylococcus aurous strain when compared with the reference antibacterial drugs ampicillin and vancomycin. It also showed a specific activity against the Escherichia coli strain with inhibition zone values of 15 mm and MIC value of 65 mg/mL, which are equipotent to the reference drugs. Compound 13 significantly inhibited the growth of the Candida albicans strain with an inhibition zone value of 11 mm, and surpassed the efficacy of the reference antibacterial drug ampicillin. It was worthwhile to note that compound 15 showed moderate activities against Staphylococcus aurous, Escherichia coli, and Candida albicans strains with inhibition zone values in the range of 10-12 mm and MIC values of 80, 75, and 77 mg/mL, respectively. On the other hand, the synthesized quinazoline-2,4-dione derivatives 14a and 14b with oxadiazole and thiadiazol moieties at the 1-and 3-poisitions of the quinazoline backbone could effectively inhibit the growth of Staphylococcus aurous and Candida albicans strains in which both compounds exhibited moderate activity against Staphylococcus aurous with inhibition zone values of 12 and 13 mm and MIC values of 70 and 75 mg/mL, respectively. The Candida albicans strain was quite sensitive to compound 14a with an inhibition zone value of 12 mm, which was more effective than the standard drug ampicillin, as depicted in Table 1.
In this context and concerning the structure-activity relationship (SAR), the synthesized compounds 13, 14a, 14b, and 15 indicated that the integration of triazole (compound 13), oxadiazole (compound 14a), thiadiazole (compound 14b), or oxazole (compound 15) across different linkers at position N-1 and N-3 of the quinazoline nucleus was essential for antimicrobial activity. It is worthwhile mentioning that our previous work that included the synthesis and SAR of a novel series of 1,3-disubistituted quinoline derivatives [49] concluded the same results. Therefore, molecular hybridization via incorporating these moieties (triazole, oxadia-zole, thiadiazole or oxazole) and others in the 1,3-positions of the quinoline or quinazoline nucleus might be thought to attain more potent antibacterial compounds.

Experimental
General Methods: All solvents and reagents were of analytical grade. The reactions were monitored by aluminum oxide thin layer chromatography TLC (silica gel 60F254, Merck, Darmstadt, Germany). Melting points were determined and were uncorrected on a melting point apparatus (Stuart Scientific, Stone, Staffordshire, UK). Mass spectra were measured on a GC MS-QP 1000 EX Mass Spectrometer (Shimadzu, Tokyo, Japan). NMR spectra were carried out with a 300 MHz at Cairo University with 400 MHz at Ulm University, Germany. Chemical shifts (δ) are reported in parts per million (ppm) relative to the respective solvent as the internal standard and the standard abbreviations were used. The required starting material 1 was prepared by adopting the earlier reported procedures [40]. Compounds 2, 3, 4, and 5 were known before and explored in scientific research [56,57].
The spectral data for the target compounds are shown in the Electronic Supplementary Materials.

2,2 -(2,4-Dioxoquinazoline-1,3(2H,4H)-diyl)diacetonitrile (4)
A mixture of 1 (1.0 g, 6.2 mmol) and potassium carbonate anhydrous (K 2 CO 3 ) (1.7 g, 12.4 mmol) in DMF (10 mL) was stirred for about 15 min at room temperature. Then, chloroacetonitrile (0.94 g, 12.4 mmol) was added. The reaction mixture was stirred for 24 h at room temperature, poured onto ice water, where the solid product was collected by filtration, washed with water, and dried to give 4 (1 g, 68%) as an off white powder; m.p. To a solution of potassium hydroxide (0.74 g, 13.2 mmol) in ethanol/water (7 mL/7 mL) were hydrazide 3 (2.0 g, 6.6 mmol) and carbon disulfide (1.0 g, 13.2 mmol) added. The reaction mixture was heated under reflux for 8 h, and subsequently, the solvent was concentrated under high vacuum before adding water and acidified with conc. hydrochloric acid. The formed solid was filtered off, washed with water, and the obtained compound was further purified by crystallizing from ethanol and dried to give 7 (1.

General Procedure for the Synthesis of 8a and 8b
A mixture of 7 (0.3 g, 0.8 mmol) and K 2 CO 3 (0.22 g, 1.6 mmol) in DMF (3 mL) was stirred for about 30 min at room temperature. Then, benzyl chloride or methyl iodide (0.8 mmol) was added. The reaction mixture was stirred for 24 h at room temperature, poured onto ice water, and the solid product was collected by filtration and the obtained compounds were further purified by crystallizing from ethanol.

General Procedure for the Synthesis of 9a and 9b
To a solution of 3 (0.2 g, 0.7 mmol) in absolute ethanol (2 mL), diethyl ethoxymethylenemalonate or ethyl ethoxymethylene cyanoacetate (1.4 mmol) was added and the reaction mixture was refluxed for 20 h. The reaction mixture was cooled to reach room temperature, poured into ice water, then the solid product was collected by filtration and washed with ethanol to give 9a and 9b, respectively.

General Procedure for the Synthesis of Compounds 12a and 12b
In a 25 mL one-necked flask, a mixture of 3 (1.0 g, 3.3 mmol) and phenylisothiocyanate or phenylisocyanate (6.6 mmol) was dissolved in absolute ethanol (10 mL). The reaction mixture was heated to reflux for 10 h and the formed solid was filtered off and washed with ethanol to give 12a or 12b. A mixture of 12b (0.4 g, 0.7 mmol) was added gradually with stirring to a solution of sodium hydroxide (2N, 5 mL). The mixture was heated to reflux for 11 h, after that, the reaction mixture was cooled and acidified with 2 N hydrochloric acid. The resulting precipitate was filtered off, washed with water, and the obtained compound was further purified by crystallizing from ethanol and dried to give 13 (0.  13.79 (br.s, 2H, 2(-SH)) ppm. 13   To a solution of 12b (0.4 g, 0.7 mmol) in methanol (4 mL), mercuric oxide (0.3 g, 1.4 mmol) was added. The resulting mixture was heated under reflux conditions for 6 h. The precipitated mercuric sulfide was filtered off and washed several times with hot methanol. On cooling, the filtrate gave a solid product, which was filtered, washed with methanol, and dried to give 14a (0.2 g, 57%) as a white solid; m.p. [160][161][162]  The 12b (0.4 g, 0.7 mmol) was added gradually and carefully with stirring to ice-cooled conc. sulfuric acid (10.5 mL) over 10 min. At 0 • C, the reaction mixture was stirred for 6 h and carefully poured onto ice water. The solid separated out was filtered off, washed with water, and the obtained compound was further purified by crystallizing from ethanol and dried to give 14b (0.

General Procedure for the Synthesis of Compounds 15 and 16
A mixture of 12a (0.4 g, 0.8 mmol) and chloroacetonitrile or ethyl bromoacetate (1.6 mmol) in (4 mL) absolute ethanol containing anhydrous sodium acetate (0.26 g, 3.2 mmol) was heated under reflux for 12 h and 16 h, respectively. The reaction mixture was cooled, diluted with water, and allowed to stand overnight. The solid precipitate was filtered off, washed with water, and the obtained compounds were further purified by crystallizing from ethanol/acetone (8:2) and dried to give 15 and 16, respectively.

Antibacterial Assay
The stock cultures of pathogens used in this study were maintained on nutrient agar slants at 4 • . The reference antibacterial drugs ampicillin and vancomycin were evaluated for their antibacterial and antifungal activities and compared with the synthesized compounds. Seventy microliters of bacterial and yeast cells (10 6 CFU/mL) of each pathogen were spread on the nutrient agar plates. The wells (6 mm diameter) were dug in the inoculated agar plates and 100 µL of the tested compound in DMSO (100 mg/mL) was added to the wells. The reference antibiotic disks (10 µg/disk of ampicillin and 30 µg/disk of vancomycin) were potted onto the surface of agar inoculated plates. Before incubation, the plates were allowed to stand for 2 h at 4 • C to allow for diffusion. Subsequently, the plates were incubated for 24 h at 37 • C, except for the yeast strain, which was incubated for 24 h at 28 • C. Next, we applied the measurement of the diameter of the inhibition zone (mm) and three replicates were averaged [58].
MIC calculation: The MIC was defined as the concentration at which the bacteria and yeast do not show visible growth with respect to the positive control. Serial dilutions were prepared of the tested compounds dissolved in DMSO. In each well of the 96 well microtiter plate, a 150 µL of double strength Mueller Hinton broth medium was loaded followed by 150 µL of the 2-fold appropriate concentration, and mixed well to obtain the final concentration. Overnight broth cultures of the tested bacterial and yeast strains prepared as an inoculum of 5% (V/V) (OD = 0.5 McFarland standard) was inoculated into the respective wells. For the positive-growth control, the same inoculum size of each test strain was inoculated in wells that did not contain any of the tested compounds. The DMSO solution was tested as the negative control. The plates were statically incubated at 37 • C for 24 h. A total of 30 µL of resazurin solution (0.18%) was added to each well to act as an electron acceptor and was reduced to a pink, red, or purple resorufin colored product by active microorganisms (i.e., the inhibition of bacterial growth was appearing as a dark blue well and the presence of growth was detected by the presence of a pink, red, or purple color).