Substitution to Position Number 2 of 4(3 H )-Quinazolinone to Create New Derivatives and to Test the Antibacterial or Antifungal Effects

: The campaign “No action today, no cure tomorrow (cid:48)(cid:48) against antimicrobial resistance proposed by the World Health Organization (WHO) has not only propeled people to take action to prevent antimicrobial resistance, but has also encouraged researchers to develop new antimicrobial agents. 4( 3H )-quinazolinone and its derivatives belong to a group of compounds with many potential applications; this study was conducted to ﬁnd new derivatives of heterocyclic 4( 3H )-quinazolinone with biological effects, contributing to research on antibacterial and antifungal compounds. Using the closed-loop method between anthranilic acid and acetic anhydride, followed by reaction with aniline derivatives, a substituted product of position 3 of 4( 3H )-quinazolinone was obtained, along with bromizing to replace the hydrogen of the methyl group in position 2 with dibromo. Heterocyclic derivatives such as imidazole, triazole, and thiazole were replaced from this dibromo product to obtain 19 derivatives. The structures of these derivatives were checked by modern methods such as IR, 1 H-NMR, and MS. The results indicated that all of the structures were as expected, so the process of creating new derivatives from 4( 3H )-quinazolinone was achieved in this study. Fourteen of the derivatives, namely 3d , 3e , 3f , 3g , 3h , 3i , 3j , 3k , 3m , 3o , 3p , 3q , 3r , and 3s , had antibacterial or antifungal effects. Among these, there were ﬁve potential derivatives: Antifungal activity was observed on A. niger by 3j and 3f (MIC: 32 µ g/mL) and 3s (MIC: 64 µ g/mL), and on C. albicans by 3f (MIC: 8 µ g/mL); antibacterial activity was observed on S. aureus by 3p (MIC: 16 µ g/mL) and 3f and 3r (MIC: 32 µ g/mL), on MRSA by 3f and 3r (MIC: 32 µ g/mL), and on E. coli by 3f (MIC: 32 µ g/mL). this, the products were puriﬁed by column chromatography with an appropriate solvent system and dried at 60 ◦ C. When substituting 2-aminothiazol bromine, potassium carbonate, potassium hydrox-ide, or sodium hydride can be used; in this study, we used potassium carbonate. The process can be summarized as follows (Scheme 4):


Introduction
While the discovery of new antimicrobials is decreasing all over the world, the level of antimicrobial resistance (AMR) is considered one of the biggest global health challenges. This situation occurs when microorganisms such as microbes, viruses, fungi, and parasites change in a way that disables or reduces the effects of drugs used in the treatment of infections. It is estimated that, by 2050, antimicrobial resistance may cause 10 million deaths globally. On World Health Day on 7 April 2011, the World Health Organization proposed actions against drug resistance via the "No action today, no cure tomorrow campaign. Besides actions for preventing antimicrobial resistance, the development of new antimicrobial compounds is considered a high priority for researchers [1][2][3].
Among the studies of biological substances, 4(3H)-quinazolinone derivatives have shown abundant biological activities, such as inhibitory effects on the central nervous system, inhibition of thromboxane A2 synthesis and platelet aggregation, reduction in blood pressure, and sedative, antispasmodic, anticonvulsant, diuretic, hypothermic, analgesic, antiulcer, antituberculosis, antibacterial, antiparasitic, antiviral, and anticancer properties [4,5]. Thus, they have attracted the attention of many scientists around the world, and the development of new antimicrobial compounds has been one of the key areas of study. 4(3H)-quinazolinone is a heterocyclic compound containing two nitrogen atoms, first synthesized by the Russian chemist Niementowski in 1895 via the cyclization reaction between anthranilic acid and formamide [6,7]. Since then, more and more studies have focused on understanding the mechanism by which the Niementowski reaction can be improved, and have found a variety of methods to synthesize heterocyclic compounds containing a nucleus of 4(3H)-quinazolinone [8,9]. Indian scientists have been pioneers in the study of 4(3H)-quinazolinone derivatives and have made great contributions to the history of this compound [10]. 4(3H)-quinazolinone and its derivatives are recognized as a group of compounds with many potential applications. Meanwhile, antibiotic resistance is currently being investigated as a major public health risk in light of the fact that we may no longer have antibiotics to fight against infectious diseases. Thus, this study was conducted in order to find new derivatives of heterocyclic 4(3H)-quinazolinone with biological effects, contributing to research on antibacterial and antifungal compounds, serving the pharmaceutical industry in the production of antibiotics in the future.
N-acetylanthranilic acid and 2-methyl-4H-3,1-benzoxazin-4-on were both synthesized by the reaction between anthranilic acid and anhydric acid. However, closed-loop products were easily hygroscopic to open rings, so the substituted 2,3 of 4(3H)-quinazolinone should be prepared using the in situ 2-methyl-4(3H)-quinazolinone method. After removal, the residual solvent was then further reacted with aniline derivatives. The reaction process can be summarized as follows (Scheme 1): The experiments were carried out in two-neck flasks. First of all, anthranilic acid was placed into a two-neck flask. Then, acetic anhydride (Ac2O) was added, heated under reflux, and then stirred. After eliminating acetic anhydride and acetic acid (AcOH) in a reduced-pressure, the mixture was transferred into another two-neck flask, then aniline and polyphosphoric acid (PPA) derivatives were added to the combination before the mixture was heated at approximately 130-140 • C. This was followed by a reaction process via thin-layer chromatography. The experiments were carried out in two-neck flasks. First of all, anthranilic acid w placed into a two-neck flask. Then, acetic anhydride (Ac2O) was added, heated und reflux, and then stirred. After eliminating acetic anhydride and acetic acid (AcOH) i reduced-pressure, the mixture was transferred into another two-neck flask, then anil and polyphosphoric acid (PPA) derivatives were added to the combination before mixture was heated at approximately 130-140 °C. This was followed by a reaction proc via thin-layer chromatography.
After completing the reaction, the combination vessel was left to cool down; then, mixture was added to the 10% alcohol solvent and heated slowly. Next, the mixture w filtered to obtain the precipitate. The precipitate was re-crystallized in 10% alcohol a bleached with activated carbon and then dried at 60 °C.
2.2.2. Synthesis of 2-dibromomethyl-3-p-substitutedphenyl-4(3H)-quinazolinone (2a-d 2-dibromomethyl-3-p-substitutedphenyl-4(3H)-quinazolinone was brominated in a methyl group (-CH3) using NBS (Br2 can also be used). NBS was chosen because i less toxic and easier to manipulate than Br2. The reaction process can be summarized follows (Scheme 2) [12]: The 2-methyl-4(3H)-quinazolinone derivative was placed into a two-neck flask co taining anhydrous chloroform, and the mixture was stirred. After this, NBS and a f drops of pyridine were added to the mixture, which was stirred continuously. The m ture was heated and stirred at 55 °C, then the reaction process was checked with TLC. T mixture after reaction was filtered to remove excess NBS, shaken with Na2S2O5 solution react with excess NBS (if any), then continued to the anhydrous stage with sodium sulf under reduced pressure. The products were purified by column chromatography with appropriate solvent system before being dried at 60 °C. Scheme 1. Synthesis of 2-methyl-3-p-substitutedphenyl-4(3H)-quinazolinone (1a-d).
After completing the reaction, the combination vessel was left to cool down; then, the mixture was added to the 10% alcohol solvent and heated slowly. Next, the mixture was filtered to obtain the precipitate. The precipitate was re-crystallized in 10% alcohol and bleached with activated carbon and then dried at 60 • C.

Synthesis of
2-dibromomethyl-3-p-substitutedphenyl-4(3H)-quinazolinone was brominated into a methyl group (-CH3) using NBS (Br 2 can also be used). NBS was chosen because it is less toxic and easier to manipulate than Br 2 . The reaction process can be summarized as follows (Scheme 2) [12]: Scheme 1. Synthesis of 2-methyl-3-p-substitutedphenyl-4(3H)-quinazolinone (1a-d) The experiments were carried out in two-neck flasks. First of all, anthra placed into a two-neck flask. Then, acetic anhydride (Ac2O) was added, reflux, and then stirred. After eliminating acetic anhydride and acetic acid reduced-pressure, the mixture was transferred into another two-neck flask and polyphosphoric acid (PPA) derivatives were added to the combinati mixture was heated at approximately 130-140 °C. This was followed by a re via thin-layer chromatography.
After completing the reaction, the combination vessel was left to cool d mixture was added to the 10% alcohol solvent and heated slowly. Next, th filtered to obtain the precipitate. The precipitate was re-crystallized in 10% bleached with activated carbon and then dried at 60 °C.

Synthesis of 2-dibromomethyl-3-p-substitutedphenyl-4(3H)-quinazol
2-dibromomethyl-3-p-substitutedphenyl-4(3H)-quinazolinone was bro a methyl group (-CH3) using NBS (Br2 can also be used). NBS was chosen less toxic and easier to manipulate than Br2. The reaction process can be su follows (Scheme 2) [12]: Scheme 2. Synthesis of 2-dibromomethyl-3-p-substitutedphenyl-4(3H)-quinazolino The 2-methyl-4(3H)-quinazolinone derivative was placed into a two-n taining anhydrous chloroform, and the mixture was stirred. After this, N drops of pyridine were added to the mixture, which was stirred continuou ture was heated and stirred at 55 °C, then the reaction process was checked w mixture after reaction was filtered to remove excess NBS, shaken with Na2S react with excess NBS (if any), then continued to the anhydrous stage with s under reduced pressure. The products were purified by column chromatog appropriate solvent system before being dried at 60 °C.

Synthesis of 2-disubstitutedmethyl-3-p-substitutedphenyl-4(3H)-quin (3a-s)
In order to replace bromine with imidazole or triazole, sodium hydri ium, or sodium ethylate can be utilized. In this study, sodium ethylate was it is possible to prepare it in situ from absolute ethanol and metal sodium [13 can be summarized as follows (Scheme 3): The 2-methyl-4(3H)-quinazolinone derivative was placed into a two-neck flask containing anhydrous chloroform, and the mixture was stirred. After this, NBS and a few drops of pyridine were added to the mixture, which was stirred continuously. The mixture was heated and stirred at 55 • C, then the reaction process was checked with TLC. The mixture after reaction was filtered to remove excess NBS, shaken with Na 2 S 2 O 5 solution to react with excess NBS (if any), then continued to the anhydrous stage with sodium sulfate under reduced pressure. The products were purified by column chromatography with an appropriate solvent system before being dried at 60 • C.

Synthesis of 2-disubstitutedmethyl-3-p-substitutedphenyl-4(3H)-quinazolinone (3a-s)
In order to replace bromine with imidazole or triazole, sodium hydride, butyl lithium, or sodium ethylate can be utilized. In this study, sodium ethylate was used because it is possible to prepare it in situ from absolute ethanol and metal sodium [13]. The process can be summarized as follows (Scheme 3): Sodium was added into a two-neck flask containing absolute ethanol (there w delay time for the sodium to dissolve) before being stirred for 30 min. Then, imida azole was added into the mixture and stirred for 60 min. In the next step, the qu none derivative was added, stirred at room temperature, and refluxed. Then, the process was checked by TLC.
After refrigerating the mixture, water was added to break down the excess ethylate, and the products in the mixture were extracted with chloroform. Then, t roform was evaporated to obtain raw products. After this, the products were pur column chromatography with an appropriate solvent system and dried at 60 °C.
When substituting 2-aminothiazol bromine, potassium carbonate, potass droxide, or sodium hydride can be used; in this study, we used potassium carbon process can be summarized as follows (Scheme 4): Quinazolinone derivatives, 2-aminothiazol, and potassium carbonate wer into a two-neck flask containing ethanol. Then, the mixture was stirred and reflux reaction was checked by TLC.
The solvent was removed from the reaction mixture; the sediment was d with chloroform and shaken with distilled water several times to remove inorga in the sediment, and then an anhydrous chloroform solution was achieved with sulfate before removing the chloroform to obtain the raw products.
The products were purified by column chromatography with an appropriate system and then dried at 60 °C.
This study aimed to generate potential compounds for the development of terial and antifungal agents, not to create a completely novel structural compoun data system of basic chemistry. Therefore, we used three kinds of spectra-IR, 1 H-NMR-to confirm whether the 4(3H)-quinazolinone derivatives created from thesis process matched the predictions.

Testing of the Antifungal and Antibacterial Effects
Antifungal effect: The diffusion method in agar was used in this study. F quinazolinone derivative was dissolved in DMSO to obtain a mixture with a conce Sodium was added into a two-neck flask containing absolute ethanol (there was some delay time for the sodium to dissolve) before being stirred for 30 min. Then, imidazole/triazole was added into the mixture and stirred for 60 min. In the next step, the quinazolinone derivative was added, stirred at room temperature, and refluxed. Then, the reaction process was checked by TLC.
After refrigerating the mixture, water was added to break down the excess sodium ethylate, and the products in the mixture were extracted with chloroform. Then, the chloroform was evaporated to obtain raw products. After this, the products were purified by column chromatography with an appropriate solvent system and dried at 60 • C.
When substituting 2-aminothiazol bromine, potassium carbonate, potassium hydroxide, or sodium hydride can be used; in this study, we used potassium carbonate. The process can be summarized as follows (Scheme 4): Sodium was added into a two-neck flask containing absolute ethanol (there w delay time for the sodium to dissolve) before being stirred for 30 min. Then, imid azole was added into the mixture and stirred for 60 min. In the next step, the q none derivative was added, stirred at room temperature, and refluxed. Then, the process was checked by TLC.
After refrigerating the mixture, water was added to break down the excess ethylate, and the products in the mixture were extracted with chloroform. Then, roform was evaporated to obtain raw products. After this, the products were pu column chromatography with an appropriate solvent system and dried at 60 °C.
When substituting 2-aminothiazol bromine, potassium carbonate, potass droxide, or sodium hydride can be used; in this study, we used potassium carbon process can be summarized as follows (Scheme 4): Quinazolinone derivatives, 2-aminothiazol, and potassium carbonate wer into a two-neck flask containing ethanol. Then, the mixture was stirred and reflu reaction was checked by TLC.
The solvent was removed from the reaction mixture; the sediment was d with chloroform and shaken with distilled water several times to remove inorga in the sediment, and then an anhydrous chloroform solution was achieved with sulfate before removing the chloroform to obtain the raw products.
The products were purified by column chromatography with an appropriat system and then dried at 60 °C.
This study aimed to generate potential compounds for the development of terial and antifungal agents, not to create a completely novel structural compoun data system of basic chemistry. Therefore, we used three kinds of spectra-IR, 1 H-NMR-to confirm whether the 4(3H)-quinazolinone derivatives created from thesis process matched the predictions.

Testing of the Antifungal and Antibacterial Effects
Antifungal effect: The diffusion method in agar was used in this study. F quinazolinone derivative was dissolved in DMSO to obtain a mixture with a conce of 2048 μg/mL. Next, a volume of approximately 10 μL of the mixture was add hole on the agar plates with a 3 mm diameter, which were cultured with C. albic niger. Then, the agar plates were incubated at 35-37 °C for C. albicans and at 30 Quinazolinone derivatives, 2-aminothiazol, and potassium carbonate were added into a two-neck flask containing ethanol. Then, the mixture was stirred and refluxed. The reaction was checked by TLC.
The solvent was removed from the reaction mixture; the sediment was dissolved with chloroform and shaken with distilled water several times to remove inorganic salts in the sediment, and then an anhydrous chloroform solution was achieved with sodium sulfate before removing the chloroform to obtain the raw products.
The products were purified by column chromatography with an appropriate solvent system and then dried at 60 • C.
This study aimed to generate potential compounds for the development of antibacterial and antifungal agents, not to create a completely novel structural compound for the data system of basic chemistry. Therefore, we used three kinds of spectra-IR, MS, and 1 H-NMR-to confirm whether the 4(3H)-quinazolinone derivatives created from this synthesis process matched the predictions.

Testing of the Antifungal and Antibacterial Effects
Antifungal effect: The diffusion method in agar was used in this study. First, the quinazolinone derivative was dissolved in DMSO to obtain a mixture with a concentration of 2048 µg/mL. Next, a volume of approximately 10 µL of the mixture was added into a hole on the agar plates with a 3 mm diameter, which were cultured with C. albicans or A. niger. Then, the agar plates were incubated at 35-37 • C for C. albicans and at 30 • C for A. niger for 24-48 h. Reading the results: For the control hole containing DMSO, it was recorded that it did not inhibit the growth of fungi. In contrast, the derivative was recorded as being resistant to fungi with the appearance of antifungal rings [14].
Antibacterial effect: The procedure was similar to that of testing the antifungal effects, except for incubation time, which was set at 16-18 h (24 h if the bacterium was MRSA) [15][16][17].

Determination of the MIC of Those Derivatives with Antifungal Antibacterial Effects
The derivatives that revealed antifungal or antibacterial effects were further tested for their minimal inhibitory concentration (MIC). The MIC was determined by the semiquantitative dilution test. The results are expressed as the minimum concentration of the test substance (µg/mL) that inhibited the growth of fungi or bacteria [14,16,18].
The fungi suspension was spread onto agar medium plates to reach a fungi density of 10 4 and 10 3 CFU/mL for A. niger and C. albicans, respectively. After this, the agar plates were incubated at 37 • C for C. albicans and at 30 • C for A. niger for 24-48 h, and then holes were made on the agar plates.
The bacteria suspension was spread onto agar medium plates to achieve a bacteria density of 10 4 CFU/mL for each species-E. coli, P. aeruginosa, B. subtilis, S. faecalis, S. aureus, methicillin-resistant S. aureus (MRSA). Similarly to the procedure of the antifungal test, the agar plates were incubated at 37 • C for 16-18 h (24 h if the bacterium was MRSA), and then holes were made on the agar plates.
The derivative was dissolved in DMSO and diluted by DMSO at concentrations of 1024, 512, 256, 128, 64, 32, 16,8,4, and 2 µg/mL. Then, each concentration solution was added into every separated hole on the agar plates as above.
Reading the results: The hole with the lowest concentration was the MIC.
Antibacterial effect: The procedure was similar to that of testing the antif fects, except for incubation time, which was set at 16-18 h (24 h if the bacter MRSA) [15][16][17].

Determination of the MIC of Those Derivatives with Antifungal Antibacterial Effects
The derivatives that revealed antifungal or antibacterial effects were furth for their minimal inhibitory concentration (MIC). The MIC was determined by t quantitative dilution test. The results are expressed as the minimum concentrati test substance (μg/mL) that inhibited the growth of fungi or bacteria [14,16,18].
The fungi suspension was spread onto agar medium plates to reach a fung of 10 4 and 10 3 CFU/mL for A. niger and C. albicans, respectively. After this, the ag were incubated at 37 °C for C. albicans and at 30 °C for A. niger for 24-48 h, and th were made on the agar plates.
The bacteria suspension was spread onto agar medium plates to achieve a density of 10 4 CFU/mL for each species-E. coli, P. aeruginosa, B. subtilis, S. faeca reus, methicillin-resistant S. aureus (MRSA). Similarly to the procedure of the an test, the agar plates were incubated at 37 °C for 16-18 h (24 h if the bacterium was and then holes were made on the agar plates.
Reading the results: The hole with the lowest concentration was the MIC.

Antifungal and Antibacterial Tests of the Derivatives
Upon testing 19 derivatives, three revealed antifungal activity with two strains and 14 (including the three derivatives mentioned) revealed antibacterial activity with five strains. None of the derivatives were recorded as having antibacterial activity against B. subtilis PY79 (Table 4).

MIC of Derivatives with Antifungal and Antibacterial Effects
Antifungal: On A. niger at a MIC of 32 µg/mL (3f and 3j) and at 64 µg/mL (3s). On C. albicans, only 3f at 8 µg/mL (Table 5); compared to the MIC on Candida spp. For flucytosine (an antifungal agent), the MIC was found to be at a sensitive level [14].  of derivatives 3d, 3e, 3f, 3g, 3h, 3i, 3j, 3k, 3m, Antibacterial: On S. aureus at a MIC of 16 µg/mL (3p) and at 32 µg/mL (3f and 3r), and on MRSA at 32 µg/mL (3f and 3r) ( Table 5); compared to the MIC on Staphylococcus spp. for some antibiotics from cephem (cefazoline, cefotaxime, ceftazidime, ceftriaxone, and cefaclor) and the glycopeptide group (vancomycin), this MIC was at an intermediate level, but, compared to the others from the cephem group (cefmetazole, cefoperazone, and cefotetan) and the aminoglycoside group (amikacin, kanamycin), the MIC was found to be at a sensitive level [16]. Furthermore, 3f on E. coli at a MIC of 32 µg/mL; compared to the MIC on Enterobacteriaceae for penicillin antibiotics (piperacillin and carbenicillin) and the cephem group (cefoperazone, cefotaxim, and ceftriaxone), this MIC was found to be at a sensitive level [16].

Discussion
From the results of this research, it was found that, although the 13 C NMR spectrum has not been measured yet, the results of the three kinds of spectra of IR, MS, and 1 H-NMR were strong enough for the authors to confirm that the substances created from this synthesis process matched the predictions. Therefore, other researchers can utilize this process to synthesize the 4(3H)-quinazolinone derivatives mentioned in the title on a larger scale.
In particular, in line with the goals of this study, 14 substances were revealed to possess antibacterial and antifungal effects, in which there were five potential derivatives with good MICs. Hence, other researchers could expand our pilot study to other bacterial or fungal strains to obtain more data. By the results of this study, the derivatives 3a-s showed different effects on bacteria and fungi because of the main difference of R1 or R2 in the 2-disubstitutedmethyl functional group of 4(3H)-quinazolinone derivatives, in which we should focus on five potential derivatives (3f, 3j, 3p, 3r, 3s). Among them, there were four derivatives containing R1 = R2 (3f: R1 = R2 = Thi; 3j: R1 = R2 = Tri; 3p: R1 = R2 = Tri; 3s: R1 = R2 = Imi), which means the C in the 2-disubstitutedmethyl functional group is not the C* (asymmetric carbon atom), and there is no C* in these compounds, so we except the chirality effects to five potential derivatives in this study.
Additionally, there are some main ways that the antibiotics act on bacteria: They inhibit DNA synthesis, RNA synthesis, cell wall synthesis, and protein synthesis [19], meaning that the antibiotics must cross the bacterial cell membrane to achieve such inhibition. The boundary of the cell membranes behaves as a lipid-like barrier against the passage of many foreign organic compounds. Numerous drugs diffuse across the cell boundary due to the fact that they are lipid soluble, and the rates of transfer are, in general, related to the relative lipid/water partition coefficients of the molecules and the almost lipid-insoluble or ionized forms of drugs that can difficultly diffuse across these boundaries [20]. In addition, some studies have confirmed the correlation of bacterial cell membranes with antimicrobial agents, in which the lipid composition of the bacterial membrane could affect the effect of antibacterial agents, indicating the importance of the lipid composition of bacterial membranes in determining the susceptibility of an organism to the action of certain antimicrobial agents [21]. Based on the results of this study, most R1 = R2 = heterocyclic compounds (3f: R1 = R2 = Thi; 3j: R1 = R2 = Tri; 3p: R1 = R2 = Tri; 3s: R1 = R2 = Imi) had better antibacterial effects than R1 = halogen compounds. Because R1 = R2 = heterocyclic compounds were well dissolved in the lipid medium, their permeability through cell membranes was better than that of R1 = halogen compounds. This important property helps active substances penetrate fungal and bacterial cells, leading to the enhancement of their antibacterial and antifungal effects, in which the difference between 3f and other substances is the thiazole heterocycle, consisting of 1 N and 1 S. Thus, it can be preliminarily recognized that different heterocyclic elements are able to increase the capacity of cell membrane permeability. Therefore, the antibacterial and antifungal effects can be optimized in further research by focusing on substance 3f or by increasing the solubility of potential substances in lipids by using other heterocycles as agent replacements, with particular attention paid to heterocycles containing N and S.

Conclusions
Using the closed-loop method between anthranilic acid and acetic anhydride, followed by a reaction with aniline derivatives, a substituted product of position 3 of 4(3H)quinazolinone was obtained, along with bromizing to replace the hydrogen of the methyl group in position 2 with dibromo. Heterocyclic derivatives such as imidazole, triazole, and thiazole were replaced by this dibromo product to obtain 19 derivatives. The structures of these derivatives were checked using modern methods such as IR, 1 H-NMR, and MS, the results of which indicated that all of the structures were as expected, so the process of creating new derivatives from 4(3H)-quinazolinone was achieved in this study.
In terms of antifungal activity, 3j and 3s revealed such activity on A. niger at a MIC of 32 µg/mL (3j) and 64 µg/mL (3s), while 3f was active on both A. niger and C. albicans at a MIC of 32 µg/mL and 8 µg/mL.