Design, Synthesis, and Bioactivity of Novel Quinazolinone Scaffolds Containing Pyrazole Carbamide Derivatives as Antifungal Agents

In this study, 32 novel quinazolinone-scaffold-containing pyrazole carbamide derivatives were designed and synthesized in a search for a novel fungicide against Rhizoctonia solani. Single-crystal X-ray diffraction of 3-(difluoromethyl)-N-(2-((6,7-difluoro-4-oxoquinazolin-3(4H)-yl)methyl)phenyl)-1-methyl-1H-pyrazole-4-carboxamide (6a11) confirmed the structure of the target compounds. The in vitro antifungal activity of the target compounds against R. solani was evaluated at 100 µg/mL. The structure–activity relationship analysis results revealed that antifungal activity was highest when the substitution activity was at position 6. Moreover, the position and number of chlorine atoms directly affected the antifungal activity. Further in vitro bioassays revealed that 6a16 (EC50 = 9.06 mg/L) had excellent antifungal activity against R. solani that was higher than that of the commercial fungicide fluconazole (EC50 = 12.29 mg/L) but lower than that of bixafen (EC50 = 0.34 mg/L). Scanning electron microscopy), 7.33 (SEM) revealed that N-(2-((6,8-dichloro-4-oxoquinazolin-3(4H)-yl)methyl)phenyl)-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide (6a16) also affected the mycelial morphology. The findings revealed that molecular hybridization was an effective tool for designing antifungal candidates. Meanwhile, pyrazolecarbamide derivatives bearing a quinazolinone fragment exhibited potential antifungal activity against R. solani.


Introduction
Rhizoctonia solani Kunh can infect the leaves and roots of various crops [1,2], causing significant yield loss. Specifically, R. solani causes sheath blight in rice, leading up to 40% yield loss [3,4]. Currently, chemical pesticides remain the most effective strategy for controlling rice sheath blight, because of the lack of rice sheath blight-resistant varieties. However, the abuse of chemical pesticides leads to pesticide resistance by pathogenic fungi. Therefore, new pesticide formulations that are more effective and more friendly to the environment need to be continuously developed.
Heterocyclic compounds are the largest class of organic compounds and play a significant role in the development of pesticides, irrespective of whether they are natural or synthetic [5,6]. New super-efficient pesticides, most of which contain heterocyclic compounds, are continuously being developed. Nitrogen-containing heterocyclic compounds, such as pyrazole and quinazolinone, have been found to have good anti-fungal activity.
Notably, pyrazole amides have become a hot spot in fungicide research because of their unique mechanism of action, safety, and efficiency [7]. Pesticide companies have developed pyrazole amide fungicides, such as isopyrazam [8], benzovindiflupyr [9], sedaxane [10], bixafen [11], and fluxapyroxad [12] (Figure 1), whose common feature is their connection by benzene rings as bridges. Quinazolinone is a benzopyrimidine heterocyclic compound that is the backbone structure of various alkaloids and drugs [13,14]. It has been widely used in the fields of medicine and pesticides because of its various excellent properties, such as its antifungal [15,16], antibacterial [17], antitumor [18], and antiviral activities [19,20]. For example, quinazolinone derivatives have been successfully developed into a commercial fungicide, fluconazole, which is used to control various fungal diseases caused by basidiomycetes, deuteromycetes, and discomycetes [21,22]. Albaconazole [23], which contains quinazolin-4-one, also exhibits a broad-spectrum antifungal activity ( Figure 2). A molecular hybrid is a combination of two or more independently acting pharmacophores that are covalently linked [24]. It can be achieved by linking or by framework integration of two or more molecules to form one molecule with increased pharmacological activity. In this study, a quinazolinone structural unit and a pyrazole-containing active fragment were combined into one molecular structure. The two were connected using a benzene ring and used to design and synthesize a series of quinazolinone-containing pyrazole carboxamide derivatives ( Figure 3). The molecular structure of the hybrid molecule was determined using 1 H NMR, 13 C NMR, and HRMS, followed by a preliminary Quinazolinone is a benzopyrimidine heterocyclic compound that is the backbone structure of various alkaloids and drugs [13,14]. It has been widely used in the fields of medicine and pesticides because of its various excellent properties, such as its antifungal [15,16], antibacterial [17], antitumor [18], and antiviral activities [19,20]. For example, quinazolinone derivatives have been successfully developed into a commercial fungicide, fluconazole, which is used to control various fungal diseases caused by basidiomycetes, deuteromycetes, and discomycetes [21,22]. Albaconazole [23], which contains quinazolin-4-one, also exhibits a broad-spectrum antifungal activity ( Figure 2). ane [10], bixafen [11], and fluxapyroxad [12] (Figure 1), whose common feature connection by benzene rings as bridges. Quinazolinone is a benzopyrimidine heterocyclic compound that is the b structure of various alkaloids and drugs [13,14]. It has been widely used in the medicine and pesticides because of its various excellent properties, such as its an [15,16], antibacterial [17], antitumor [18], and antiviral activities [19,20]. For e quinazolinone derivatives have been successfully developed into a commercial fu fluconazole, which is used to control various fungal diseases caused by basidiom deuteromycetes, and discomycetes [21,22]. Albaconazole [23], which contains qui 4-one, also exhibits a broad-spectrum antifungal activity ( Figure 2). A molecular hybrid is a combination of two or more independently acting cophores that are covalently linked [24]. It can be achieved by linking or by fra integration of two or more molecules to form one molecule with increased pharm ical activity. In this study, a quinazolinone structural unit and a pyrazole-contai tive fragment were combined into one molecular structure. The two were connect a benzene ring and used to design and synthesize a series of quinazolinone-co pyrazole carboxamide derivatives ( Figure 3). The molecular structure of the hybr cule was determined using 1 H NMR, 13 C NMR, and HRMS, followed by a pre A molecular hybrid is a combination of two or more independently acting pharmacophores that are covalently linked [24]. It can be achieved by linking or by framework integration of two or more molecules to form one molecule with increased pharmacological activity. In this study, a quinazolinone structural unit and a pyrazole-containing active fragment were combined into one molecular structure. The two were connected using a benzene ring and used to design and synthesize a series of quinazolinone-containing pyrazole carboxamide derivatives ( Figure 3). The molecular structure of the hybrid molecule was determined using 1 H NMR, 13 C NMR, and HRMS, followed by a preliminary screening of its antifungal activity in vitro. Subsequent physiological assays of compounds with high antifungal activity against R. solani, which causes sheath blight in rice, were further conducted.
screening of its antifungal activity in vitro. Subsequent physiological assays pounds with high antifungal activity against R. solani, which causes sheath bligh were further conducted.

Instruments and Chemicals
1 H and 13 C NMR spectra were recorded in DMSO-d6 using 400 and 100 MHz photometers (Bruker BioSpin GmbH, Rheinstetten, Germany), respectively, wh resolution mass spectrometry (HRMS) was performed using Thermo Scientific Q (Thermo Fisher Scientific, Massachusetts, USA). The X-ray crystallographic data w lected and processed using a D8 Quest X-ray diffractometer (Bruker BioSpin Rheinstetten, German). All solvents were distilled and dried using standard meth fore use.

Materials and Methods
2.1. Chemistry 2.1.1. Instruments and Chemicals 1 H and 13 C NMR spectra were recorded in DMSO-d 6 using 400 and 100 MHz spectrophotometers (Bruker BioSpin GmbH, Rheinstetten, Germany), respectively, while highresolution mass spectrometry (HRMS) was performed using Thermo Scientific Q Exactive (Thermo Fisher Scientific, Waltham, MA, USA). The X-ray crystallographic data were collected and processed using a D8 Quest X-ray diffractometer (Bruker BioSpin GmbH, Rheinstetten, German). All solvents were distilled and dried using standard methods before use.

General Procedure for the Preparation of the Target Compounds 6a 1 -a 32
Substituted quinazolinone (1 mmol, 5a 1 -a 32 ) was added to 10 mL DMF and stirred to dissolve. KOH (59 mg, 1.05) was added and left to react for 40 min, after which 4 (299 mg, 1 mmol) was added. The reaction was carried out at room temperature for 1-3 h. The reaction was detected by TLC, followed by the addition and stirring of 20 mL of saturated ammonium chloride solution for 5 min. It was then extracted with dichloromethane (20 mL × 3), dried over anhydrous sodium sulfate, filtered, concentrated, and purified (Scheme 3).
formamidine acetate (20 mmol) in ethylene glycol monomethyl ether and the reaction mixture was subsequently stirred at 95-130 °C. The mixture was then poured into cold water, thereby precipitating a large amount of solid. The crude product was subsequently obtained by filtration and was dissolved in hot 10% NaOH solution, heated for 5-6 min with charcoal and filtered, and the clear solution was subsequently neutralized (pH = 7) using 1N HCl. The precipitated crystals were filtered out, washed with cooled water, and dried to obtain the quinazolin-4-ones (Scheme 2, Supplementary Materials).

General Procedure for the Preparation of the Target Compounds 6a1-a32
Substituted quinazolinone (1 mmol, 5a1-a32) was added to 10 mL DMF and stirred dissolve. KOH (59 mg, 1.05) was added and left to react for 40 min, after which 4 (299 m 1 mmol) was added. The reaction was carried out at room temperature for 1-3 h. Th reaction was detected by TLC, followed by the addition and stirring of 20 mL of saturate ammonium chloride solution for 5 min. It was then extracted with dichloromethane ( mL×3), dried over anhydrous sodium sulfate, filtered, concentrated, and purified (Schem 3).  13  3     3     13

In Vitro Antifungal Assay
R. solani was the test strain. It was provided by the Guizhou Institute of Plant Protection. In this study, the in vitro antifungal activity of the target compound 6a 1 -a 32 against R. solani was screened using the mycelial growth rate method [27,28]. The commercial fungicides fluconazole and bixafen were selected as the positive controls. R. solani was inoculated on potato dextrose agar (PDA) plates and grown in biochemical incubators at 28 ± 1 • C for 2 days. The newly grown mycelia were used to determine the antifungal activity. The tested compounds were dissolved in DMSO to prepare 10 mg/mL stock solutions before mixing with PDA. The PDA containing the test compounds at a concentration of 100 mg/L was then poured into sterile Petri dishes for primary screening. A data processing system (DPS, V9.50) was used for statistical analysis of the test data, and the significant differences were determined using Duncan's new multiple range method. The EC 50 values and 95% confidence limits were calculated after testing the inhibition rates, based on Duncan's new multiple range method. The inhibition rate of the potent compounds was further tested and the corresponding EC 50 values were calculated using DPS. The relative inhibitory rates of the potent compounds were then calculated using the following equation: where C is the colony diameter of the control (mm), T is the colony diameter of the treatment (mm), and 5 is the diameter of the mycelium disks.

SEM Observations
SEM observations of R. solani hyphae were conducted following the reported methods [29,30]. Mycelium disks with a diameter of 5 mm were taken from the edge of the PDA medium containing 12.29 mg/L of 6a 16 and were incubated for 2 days at 28 ± 1 • C. Mycelium disks from PDA with 0.1% DMSO were used as controls. The samples were then fixed at 4 • C using 2.5% glutaraldehyde for 1 day and were subsequently rinsed thrice with 0.1 M phosphate buffer for 15 min. The samples were then fixed with 1% OsO 4 solution for 1 h and then dehydrated in 10%, 30%, 50%, 70%, 90%, and 100% ethanol at 10 min intervals. Gold coating of the samples was then carried out after drying at the critical point, followed by SEM observations.

Chemistry
Hybrid molecules are defined as chemical entities with two or more structural domains having different biological functions and dual activity, indicating that a hybrid molecule acts as two distinct pharmacophores. Hybrid antifungal molecules 6a 1 -a 32 , based on the conjugation of quinazolinone to pyrazolecarbamide, were designed and synthesized. Schemes 1-3 show the synthetic route of target compounds 6a 1 -a 32 . First, 3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxylic acid 1 was reacted with oxalyl chloride to obtain pyrazole-4-carbonyl chloride 2, a light yellow, oily liquid [25]. Intermediate 3 (a white product) was obtained by reacting 2 with o-aminobenzyl alcohol in a saturated sodium carbonate solution. Intermediate 3 was subsequently chlorinated by dichlorosulfoxide at room temperature to yield intermediate 4, a white solid (Scheme 1). Optimized preparative procedures for all quinazolin-4-ones 5a 1 -a 32 were conducted following László Örfi [26]. Anthranilic acid or its substituted derivatives were mixed with formamidine acetate in ethylene glycol monomethyl ether at 95-130 • C to obtain quinazolin-4-ones (Scheme 2). The target compounds of quinazolinone-scaffold-containing pyrazole carboxamides were finally obtained by reacting intermediate 4 with different substituted quinazolin-4-ones 5a 1 -a 32 in DMF under alkaline conditions (Scheme 3) . The structures of all key intermediates and target compounds were confirmed using 1 H and 13 C NMR and HRMS, and their spectra data are shown in the Supplementary Materials.

X-ray Diffraction
To further validate the structure of the target compounds, the structure of 6a 11 was further identified by X-ray diffraction studies (Figure 4).

In Vitro Antifungal Assay
Tables 1 and 2 outline the preliminary in vitro antifungal activities of the 32 target compounds at 100 mg/L. Most of the target compounds exhibited some degree of antifungal activity, with inhibition rates ranging between 13.63% and 100% against R. solani (Table 1). Notably, compounds 6a 24 and 6a 29 showed good antifungal activity against R. solani, with inhibition rates of 70.26% and 70.53%, respectively. Nonetheless, these rates were lower than those of fluconazole (100%) and bixafen (100%). Hybrid antifungal molecule 6a 16 exhibited the best antifungal activity against R. solani, matching that of fluconazole and bixafen.
The EC 50 value of 6a 16 was further tested in light of its good inhibitory characteristic. The EC 50 of 6a 16 , fluconazole, and bixafen were 9.06, 12.29, and 0.34 mg/L, respectively ( Table 2), suggesting that the inhibitory activity of 6a 16 against R. solani was comparable to that of fluconazole, but worse than that bixafen.  Figure 5 shows the results of the structure-activity relationship analysis. The trend of the inhibitory activity against R. solani was highest when the quinazolinone of the target compounds was substituted with a bromine atom at substitution site 6. Notably, the same trend of antifungal activity was maintained when F-, Cl-, and CH3O-were substituted at different sites with a bromine atom. The fungal activity inhibition trend was 6-CH3O > 6-Cl > 6-Br-> 6-F > 6-CH3 when the substitution site was at position 6. In addition, the number and position of the substituent atoms affected the antifungal activity. For example, the structural differences of 6a12, 6a13, 6a14, 6a15, and 6a16 were at different positions and quantities of chlorine atoms, and their inhibition rates were 41.43%, 61.93%, 45.90%, 45.43%, and 100%, respectively. The antifungal activity at 6,8-diCl > 6-Cl > 7-Cl≈8-Cl > 5-Cl indicated that the position and number of chlorine atoms directly affected the antifungal activity. The EC50 value of 6a16 was further tested in light of its good inhibitory characteristic. The EC50 of 6a16, fluconazole, and bixafen were 9.06, 12.29, and 0.34 mg/L, respectively ( Table 2), suggesting that the inhibitory activity of 6a16 against R. solani was comparable to that of fluconazole, but worse than that bixafen.

Scanning Electron Microscopy (SEM) of the Hyphae Morphology upon Treatment of R. solani with Compound 6a16
The morphology of fungi treated with 6a16 changed significantly ( Figure 6). Notably, the mycelia of the control group were uniform in thickness, smooth, full on the surface, and well extended ( Figure 6A). However, the mycelia appeared to fold and collapse after treatment with 9.06 mg/L and 18.12 mg/L of compound 6a16 (Figure 6C).  The morphology of fungi treated with 6a 16 changed significantly ( Figure 6). Notably, the mycelia of the control group were uniform in thickness, smooth, full on the surface, and well extended ( Figure 6A). However, the mycelia appeared to fold and collapse after treatment with 9.06 mg/L and 18.12 mg/L of compound 6a 16 ( Figure 6C).

Discussion
The nucleophilic substitution of quinazolinone can result in N-and O-substitutions [31,32]. In this study, the quinazolinones 5a1-a32 were treated with chlorinated intermediate 4 in the presence of potassium hydroxide in DMF (Scheme 3). Of note, substituting quinazolin-4-ones 5a1-a32 led to the exclusive formation of N-substituted quinazolines, with no detection of O-substituted isomers. The single-crystal X-ray diffraction of compound 6a11 further showed that the target compound was an N-substituted quinazoline. Figure 4 shows the crystal structure of 6a11, whose deposition number is CCDC 2218016.
Hybrid molecules are defined as chemical entities with two or more structural domains having different biological functions and dual activity, indicating that a hybrid molecule acts as two distinct pharmacophores [24]. Hybrid molecules could explore new lead compounds. Highly selective inhibitors of human α-1,3-Fucosyltransferase and acetylcholine esterase (AChE) were produced by this strategy [33,34]. In present paper, a quinazolinone structural unit and a pyrazole-containing active fragment were hybridized to create highly reactive molecules. We also obtained hybrid 6a16 with good antifungal activity. Therefore, molecular hybridization, based on the conjugation of quinazolinone to pyrazolecarbamide, is a useful approach for designing high antifungal candidates.

Conclusions
In this study, 32 novel pyrazolecarbamide derivatives bearing quinazolinone scaffolds were successfully designed, synthesized, and characterized in detail using 1 H-NMR, 13 C-NMR, and HRMS. The preliminary results of fungicidal bioassays revealed that some of the target compounds exhibited certain inhibitory activities against R. solani. Notably, compared with the commercial fungicide fluconazole, compound 6a16 exhibited excellent antifungal activities against R. solani by affecting the mycelial morphology. The results of this study collectively suggest that 6a16 is a lead compound against R. solani and should be further explored to enhance its utility and application. Figure 6. Scanning electron micrographs of R.solani hyphae of the control group (A), hyphae exposed to 6a 16 at a concentration of 9.06 mg/L (B), and hyphae exposed to 6a 16 at a concentration of 18.12 mg/L (C).

Discussion
The nucleophilic substitution of quinazolinone can result in Nand O-substitutions [31,32]. In this study, the quinazolinones 5a 1 -a 32 were treated with chlorinated intermediate 4 in the presence of potassium hydroxide in DMF (Scheme 3). Of note, substituting quinazolin-4ones 5a 1 -a 32 led to the exclusive formation of N-substituted quinazolines, with no detection of O-substituted isomers. The single-crystal X-ray diffraction of compound 6a 11 further showed that the target compound was an N-substituted quinazoline. Figure 4 shows the crystal structure of 6a 11 , whose deposition number is CCDC 2218016.
Hybrid molecules are defined as chemical entities with two or more structural domains having different biological functions and dual activity, indicating that a hybrid molecule acts as two distinct pharmacophores [24]. Hybrid molecules could explore new lead compounds. Highly selective inhibitors of human α-1,3-Fucosyltransferase and acetylcholine esterase (AChE) were produced by this strategy [33,34]. In present paper, a quinazolinone structural unit and a pyrazole-containing active fragment were hybridized to create highly reactive molecules. We also obtained hybrid 6a 16 with good antifungal activity. Therefore, molecular hybridization, based on the conjugation of quinazolinone to pyrazolecarbamide, is a useful approach for designing high antifungal candidates.

Conclusions
In this study, 32 novel pyrazolecarbamide derivatives bearing quinazolinone scaffolds were successfully designed, synthesized, and characterized in detail using 1 H-NMR, 13 C-NMR, and HRMS. The preliminary results of fungicidal bioassays revealed that some of the target compounds exhibited certain inhibitory activities against R. solani. Notably, compared with the commercial fungicide fluconazole, compound 6a 16 exhibited excellent antifungal activities against R. solani by affecting the mycelial morphology. The results of this study collectively suggest that 6a 16 is a lead compound against R. solani and should be further explored to enhance its utility and application.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cimb44110380/s1. Author Contributions: Z.L. and W.Y. conceived and designed the paper. Z.L., H.L., X.B. and X.G. contributed to the synthesis, purification, and characterization of all compounds. J.Y. and Q.P. performed the biological activity research. Z.L. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.