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Article

The Synthesis and Biological Evaluation of 2-(1H-Indol-3-yl)quinazolin-4(3H)-One Derivatives

by
Elena Y. Mendogralo
1,*,
Larisa Y. Nesterova
1,2,
Ekaterina R. Nasibullina
1,
Roman O. Shcherbakov
1,
Alexander G. Tkachenko
1,2,
Roman Y. Sidorov
1,2,
Maxim A. Sukonnikov
3,
Dmitry A. Skvortsov
3 and
Maxim G. Uchuskin
1
1
Department of Chemistry, Perm State University, Bukireva St. 15, 614990 Perm, Russia
2
Institute of Ecology and Genetics of Microorganisms, Ural Branch of the Russian Academy of Sciences, Goleva St. 13, 614081 Perm, Russia
3
Department of Chemistry, M.V. Lomonosov Moscow State University, Leninskie Gory 1-3, 119991 Moscow, Russia
*
Author to whom correspondence should be addressed.
Molecules 2023, 28(14), 5348; https://doi.org/10.3390/molecules28145348
Submission received: 16 June 2023 / Revised: 3 July 2023 / Accepted: 8 July 2023 / Published: 11 July 2023
(This article belongs to the Section Organic Chemistry)
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Abstract

:
The treatment of many bacterial diseases remains a significant problem due to the increasing antibiotic resistance of their infectious agents. Among others, this is related to Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA) and Mycobacterium tuberculosis. In the present article, we report on antibacterial compounds with activity against both S. aureus and MRSA. A straightforward approach to 2-(1H-indol-3-yl)quinazolin-4(3H)-one and their analogues was developed. Their structural and functional relationships were also considered. The antimicrobial activity of the synthesized compounds against Mycobacterium tuberculosis H37Rv, S. aureus ATCC 25923, MRSA ATCC 43300, Candida albicans ATCC 10231, and their role in the inhibition of the biofilm formation of S. aureus were reported. 2-(5-Iodo-1H-indol-3-yl)quinazolin-4(3H)-one (3k) showed a low minimum inhibitory concentration (MIC) of 0.98 μg/mL against MRSA. The synthesized compounds were assessed via molecular docking for their ability to bind long RSH (RelA/SpoT homolog) proteins using mycobacterial and streptococcal (p)ppGpp synthetase structures as models. The cytotoxic activity of some synthesized compounds was studied. Compounds 3c, f, g, k, r, and 3z displayed significant antiproliferative activities against all the cancer cell lines tested. Indolylquinazolinones 3b, 3e, and 3g showed a preferential suppression of the growth of rapidly dividing A549 cells compared to slower growing fibroblasts of non-tumor etiology.

Graphical Abstract

1. Introduction

One of the most serious threats to human health is bacterial antibiotic resistance [1]. This problem is projected to cause 10 million deaths worldwide each year by 2050 without the development of new strategies for defeating antibiotic resistance [2]. The most commonly isolated nosocomial pathogens are Escherichia coli and Staphylococcus aureus. Of particular concern currently is methicillin-resistant S. aureus (MRSA), which is considered to be one of the most dangerous antibiotic-resistant microorganisms [3]. Another cause of a serious bacterial infection is the bacillus Mycobacterium tuberculosis. Until the outbreak of SARS-CoV-2, tuberculosis (TB) was the leading cause of death from a single infectious agent, ranking above HIV/AIDS. It is estimated that, today, about a quarter of the world’s population is infected with TB. Most people do not develop tuberculosis, and in some, the infection clears. However, without treatment, the death rate from TB disease is high (about 50%) [4]. Despite the availability of drugs for the treatment of tuberculosis, there is a need for improved therapeutic agents. Side effects, the duration of treatment, and drug resistance are the main limitations to the effectiveness of the drugs used. One of the factors affecting the resistance of some infections to common antibiotics is the role of bacterial biofilm formation. Biofilms are highly organized surface-associated communities enclosed in an extracellular polymer matrix. The bacteria in a biofilm are known to be 1000 times more resistant to antibiotics. Therefore, one of the attractive approaches to combating multidrug-resistant bacteria is the inhibition of biofilm formation.
In recent years, many biologically active compounds have been isolated from representatives of marine flora and fauna. Meridianins, a group of indole alkaloids isolated from the tunicate Aplidium meridianum [5,6], have promising anti-tuberculosis [7], antimalarial [8,9], and antitumor [10] activities. Notably, analogues of meridianin have shown powerful inhibitory activity against biofilm formation by S. aureus (MRSA) [11] and Mycobacterium smegmatis [12]. We posited that the closest structural analogues of meridianins would have the ability to control the behavior of bacteria based on the shared structural features of meridianins with quinazolinone derivatives. Quinazolinone scaffolds are among more than 150 natural alkaloids and pharmaceuticals. Quinazolinone derivatives exhibit a wide range of biological activity, with anti-inflammatory, antitumor, antipsychotic, and antibacterial activities (Figure 1) [13,14,15,16,17,18].
The different physiological processes underlying bacterial stress adaptation are under the control of indoles [19]. A family of widely distributed bacterial metabolites, termed the ‘‘indolokines”, are able to enhance E. coli persister cell formation, thereby connecting some processes, including biofilm formation, antibiotic tolerance, virulence, and others [20,21]. One of the main regulators of cell adaptive responses to stress is guanosine tetraphosphate (ppGpp), which is a bacterial alarmone participating in the dormant state formation. In this state, bacterial cells are transformed into persister cells that are tolerant to antibiotics and stresses [22]. One of the regulatory effects of ppGpp is the induction of the activity of the ribosome modulation factors that are responsible for the inhibition of protein synthesis. At the same time, it has been shown that indole is also capable of inducing one of the ribosome hibernation factors—RaiA—thereby taking part in persister cell formation [23]. We showed that ppGpp can stimulate tryptophanase induction, which is responsible for indole synthesis, thereby strengthening its own ability to form persisters [24]. Previously, we described the inhibition of alarmone synthetase enzymes, the product of which is ppGpp [25]. In turn, the inhibition of these enzymes suppressed the ability of bacterial cells to form a persister state responsible for antibiotic tolerance. Furthermore, it was shown that substituted indoles are able to bind RelSeq (p)ppGpp synthetase/hydrolase and can constitute a promising scaffold for (p)ppGpp synthetase enzyme inhibitors’ development [26].
Based on data from the literature, we hypothesized that the combination of a quinazolinone fragment with an additional azaheterocycle in a single molecule could promise a synergistic enhancement of the pharmaceutical value of these two biologically important structural subunits. A wide library of synthesized indole derivatives will make it possible to find among them those that are able to bind alarmone synthetases as the probable targets for interaction. To date, many methods have been developed for the synthesis of quinazolinones and their analogues due to their high applied significance [27]. The most common synthetic approach to quinazolin-4(3H)-ones involves the condensation of anthranilamides with aromatic aldehydes [28,29,30,31,32], ketones [33,34], alkynes [35], carboxylic acid [36], and their derivatives [37], benzyl alcohols [38], CO/aryl bromides [39], and methylarenes [40], etc. While approaches to quinazolin-4(3H)-ones are well known and capable of providing the formation of the desired framework, they suffer from certain drawbacks. Unfortunately, these methods require expensive transition metal catalysts, additives, harsh reaction conditions, and a moderate reaction scale. The most significant disadvantage of most of the described methods is associated with the limited applicability of the starting heteroaromatic substrates. Therefore, the search for a simple and efficient protocol for accessing 2-(1H-indol-3-yl)quinazolin-4(3H)-one from readily available starting substrates under simple reaction conditions is highly desirable.
Herein, we describe a straightforward approach to 2-(1H-indol-3-yl)quinazolin-4(3H)-one and their analogues based on the condensation of anthranilamides with aldehydes and an investigation of their cytotoxicity and antibacterial activity, including tests against Mycobacterium tuberculosis H37Rv, as well as their influence on the formation of biofilms. We describe the results of molecular docking studies on the ability of substituted indoles to bind to long RSH proteins, using two proteins functioning as the alarmone synthetases: RelMtb from Mycobacterium tuberculosis [41] and RelSeq from Streptococcus equisimilis as the models [26].

2. Results and Discussion

2.1. Chemistry

The starting point of our research was the search for the optimal conditions of the model reaction of commercially available 1H-indole-3-carboxaldehyde (1a) with anthranilamide (2a). Initially, we screened the reaction conditions using various Brønsted acids. The best result was achieved when the reaction mixture was stirred at reflux for 4 h in acetonitrile in the presence of p-TSA (p-toluenesulfonic acid). In this case, the yield of desired quinazolinone 3a was 33% (Scheme 1a). A thorough analysis and separation of the reaction mixture revealed the formation of indole (4a) and unsubstituted quinazolinone 5a. In other acidic conditions (amberlyst-15, CH3CN or toluene; NH4Cl or AcOH, EtOH; citric acid, toluene, etc.), various side processes were observed, which significantly decreased the yield of the desired product. Next, we found that changing the reaction initiator to Al2O3 led to the formation of 2-(1H-indol-3-yl)-2,3-dihydroquinazolin-4(1H)-one (6a) as a major product (Scheme 1b). In addition to dihydroquinazolinone 6a, some indole (4a) was isolated from the reaction mixture. The obtained results (Scheme 1a,b) indicate that the key intermediate quinazolinone 6a is unstable under used reaction conditions and can be oxidized to form the desired quinazolinone 3a or undergo degradation affording 3-unsubstituted indole (4a). Control experiments showed that the dihydroquinazolinone 6a in the EtOAc, CH2Cl2, and ethanol solutions was oxidized to key quinazolinone 3a upon standing in air. Moreover, the deformylation reaction and its related cleavage processes are well known and widely used as a key step for the preparation of natural and synthetic compounds [42,43,44,45]. Finally, the desired product 3a was obtained in a quantitative yield when the reaction was performed in N,N-dimethylacetamide (DMAC) in the presence of Na2S2O5 at 150 °C (Scheme 1c). Presumably, the true initiator of the cyclocondensation was NaHSO3, which was in situ generated via the hydrolysis of the starting Na2S2O5 [46].
With the optimized conditions in hand, we examined the substrate scope of this reaction. Initially, we performed the reaction at a 3 mmol scale and found that the yield of the desired product 3a was 86% (Scheme 2). Next, we found that anthranilamide (2a) reacted smoothly with various N-substituted 3-formylindoles 1 to form the corresponding products 3b-g in good to excellent yields. When indole-3-carboxaldehydes 1 containing easily removal groups at the nitrogen atom were used, we isolated the indolylquinazolinone 3a instead of the expected N-substituted indolylquinazolinones 3h–j. The products 3m, n, containing an aromatic substituent at the C(2) position of the indole core, were detected only in trace amounts, while quinazolinone 5a was isolated in 59 and 37% yields, respectively. The formation of quinazolinone 5a in these cases could be associated with the better leaving nature of the 2-arylindoles in the intermediate dihydroquinazolinones. The halogen atoms in the starting indole 1 or anthranilamide 2 did not have a significant influence on the yields of the desired products 3k, l, o, and 3p. A reduced yield of indolylquinazolinone 3q was associated with the partial thioamidation of the 1H-indole-3-carboxaldehyde (1a) by elemental sulfur upon the prolonged heating of the reaction mixture [47]; among the major indolylquinazolinone 3q, we isolated N,N-dimethyl-1H-indole-3-carbothioamide (7a) in a 21% yield (for details see Supplementary Materials). We also performed the reaction of indole-2-carboxaldehyde (1o) with anthranilamide (2a) and isolated the corresponding product 3r in a high yield.
Next, we expanded the scope of the developed protocol and used various carbo- and heteroaromatic aldehydes 1 as starting substrates. We found that, in all cases, the desired indolylquinazolinones 3 were isolated in high yields. The exception was the electron-deficient isonicotinaldehyde, for which the desired product 3y was obtained in a low yield. It should be noted that the use of dimethyl sulfoxide without additional reagents in the reaction of 5-methylfurfural with anthranilamide (2a) resulted in the formation of the dihydroquinazolinone 6u in a 62% yield. Product 6u, in contrast to its indole analogue 6a, was more stable and was not oxidized in the solution in air atmosphere.
We tested a series of structural analogues of anthranilamide with 1H-indole-3-carboxaldehyde (1a). We found that the reaction of aldehyde 1a with anthranilic acid hydrazide led to the corresponding product 3ab in a good yield. The reaction of salicylic amide with 1H-indole-3-carboxaldehyde (1a) unfortunately did not provide the desired product 3ac due to the predominant occurrence of the deformylation reaction. A similar result was also obtained in the case of Gewald’s thiophene. It is noteworthy that, when using thiophene-2-carbaldehyde, the product 3ad was isolated in a moderate yield. In the case of 2-aminobenzenesulfonamide and 2-amino-2-phenylacetamide, the thioamidation reaction predominated, while we did not observe the formation of the desired products 3af and 3ag. It should be noted that all the synthesized compounds were stable and fully characterized by NMR and HRMS.
Finally, we studied some reactivity of the model 2-(1H-indol-3-yl)quinazolin-4(3H)-one (3a) (Scheme 3). Initially, we checked the possibility of the functionalization of the nitrogen atom and synthesized various N-substituted indolylquinazolinones 3. The problem of the hydrolysis of the protecting group at the nitrogen atom can be solved by changing the order of the reaction steps. The treatment of indolylquinazolinone 3a with benzoyl chloride afforded 2-(1-benzoyl-1H-indol-3-yl)quinazolin-4(3H)-one (3i) in a good yield. The sequential treatment of quinazolinone 3a with NaH and methyl iodide afforded a mixture of three products in equal amounts. Along with the expected mono- 3b and disubstituted indolylquinazolinones 3ah, we observed the formation of 4-methoxy-2-(1-methyl-1H-indol-3-yl)quinazoline (3ai). (Scheme 3).
According to the literature data, it is known that some thioquinazolinones are characterized by increased antimycobacterial activity compared to that of quinazolinones [48]. To study the reactivity of the substrate 3a, we developed a method of thionation with Lawesson’s reagent (LR) and a subsequent alkylation affording the target 2-(1H-indol-3-yl)-4-(methylthio)quinazolinone (3ak) in a moderate yield over two steps (Scheme 4). An attempt to prepare an isomer of bouchardatine 3al using the Vilsmeier–Haack formylation of the 2-(1H-indol-3-yl)quinazolin-4(3H)-one (3a) was unsuccessful (Scheme 4). Instead of the expected product, 4-chloro-2-(1H-indol-3-yl)quinazoline (3am) was isolated in a moderate, nonoptimized yield. The obtained 4-chloroquinazoline 3am was treated with morpholine and the desired 4-[2-(1H-indol-3-yl)quinazolin-4-yl]morpholine (3an) was obtained in a high yield. Both observations demonstrated the possibility of accessing a wide range of 4-amino- and 4-thiosubstituted quinazolines.

2.2. In Vitro Biological Evaluation

2.2.1. Antibacterial Activity

The 32 synthesized compounds were evaluated for their antimicrobial activity against Staphylococcus aureus, Escherichia coli, Mycobacterium smegmatis, and Candida albicans. High activity against staphylococci was demonstrated by indolylquinazolinone 3k (Table 1). Compound 3k inhibited the growth of S. aureus ATCC 25923, a standard reference strain for evaluating the activity of antimicrobial compounds, showing an MIC of 3.90 μg/mL. At the same time, its MIC against the reference strain S. aureus ATCC 43300 (MRSA) was even lower and did not exceed 1 μg/mL. This is a very promising result, as MRSA is now widespread and a significant problem in human and veterinary medicine, because it is resistant to many antibacterial compounds. Additionally, we studied the activity of compound 3k against another staphylococcus species, S. epidermidis ATCC 12228. The MIC and MBC (minimum bactericidal concentration) in this case were 7.80 and 12.50 µg/mL, respectively. All of the compounds were found to be inactive against the Gram-negative bacterium E. coli. In addition to their antibacterial properties, the antimycotic activity of the synthesized substances against Candida albicans was studied (minimum fungicidal concentration—MFC). Compounds 3d, 3p, 3t, 3u, 5a, and 6u showed moderate activity against this microorganism, while substances 3k and 3ai showed more pronounced activity (MIC 7.80, 62.50 µg/mL, respectively) (Table 1). Other quinazolinone derivatives 3 were found to be inactive (See Supplementary Materials).
Mycobacteria are a special group of bacteria with unique structural and physiological features. This group includes both saprophytes and animal or human pathogens, in particular, the causative agent of tuberculosis, Mycobacterium tuberculosis. Mycobacterium smegmatis is widely used as a model microorganism for studying antimycobacterial activity. Most of the compounds were found to be inactive against M. smegmatis. Although M. smegmatis is used as a model organism for the study of M. tuberculosis, these bacteria are not identical and have their own individual characteristics. Particularly, there are significant differences in their growth rate and in the structure of their cell walls. Considering the high activity of compound 3k against Gram-positive organisms (staphylococci), we studied its activity against M. tuberculosis H37Rv using a BASTEC MGIT 960 growth system [49,50]. This compound inhibited the growth of M. tuberculosis H37Rv within 41 days at a concentration of 10 μg/mL. In the presence of 5 μg/mL of this substance, the growth of the mycobacteria was not observed for 18 days, and in the presence of 1 μg/mL, for 5 days (Table 2). Therefore, indolylquinazolinone 3k is the leading compound that is promising for further studies in the field of anti-tubercular activity.
It is worth noting that several compounds studied in this work have previously been tested for their antibacterial activity. Compounds 3k, 3t, and 3z are active against several species of Leishmania, which are intracellular parasites [8,51]. Indolylquinazolinone 3k has an antibacterial effect against M. tuberculosis, which is confirmed by our studies [7]. Compounds 3t, 3u, and 3x–z have been tested against E. coli and S. aureus, but did not show high activity [52], which is in agreement with our results. Quinazolinone 5a has an antibacterial effect against E. coli, S. aureus, Bacillus subtilis, and other bacteria [53,54], but this result is not confirmed by our studies. Perhaps this is due to the difference in the methodological approaches to the antimicrobial activity determination. In our experiments, only the antifungal activity of compound 5a was noted, which had not previously been shown.

2.2.2. Antibiofilm Activity

Biofilms are communities of microorganisms that are attached to a surface and play a significant role in bacterial persistence. This is a significant problem in the health industry and much research has been performed to reduce microbial colonization and prevent biofilm formation [55]. In this study, the ability of the most active synthesized compound 3k to prevent biofilm formation was tested. Most of the known antibiotics inhibit biofilm formation by affecting cell viability (decrease in biofilm biomass correlates with a decrease in the number of cells in a culture). We evaluated the number of living cells in a culture during biofilm formation in the presence of various concentrations of 2-(5-iodo-1H-indol-3-yl)quinazolin-4(3H)-one (3k) and the control antibiotic amikacin to ensure that the observed inhibition of biofilm formation was not the result of a decrease in the number of planktonic cells. Compound 3k was able to prevent the appearance of biofilms of staphylococci without affecting the number of cells in a culture (Figure 2b). In contrast, amikacin reduced the formation of a microbial biofilm due to its lethal effect on planktonic cells (Figure 2a).

2.2.3. Cytotoxicity Activity

Compounds 3c, f, g, k, r, and 3z showed the highest cytotoxicity, for which IC50 or IC50abs were observed in the early micromolar range (less than 10 μM) against the tested cell lines. The dependences of the number of cells on the concentration of the compounds are shown in Figure 3. It should be noted that compounds 3b, 3e, and 3g showed a preferential suppression of the growth of rapidly dividing A549 cells compared to slower growing fibroblasts of non-tumor etiology. In addition, there was a significant proportion of living cells compared to the untreated control over a wide range of high concentrations. The greatest difference was observed for compound 3e. Although this dependence can be explained, for example, by the solubility of the compounds, it is more likely to be explained by a cytostatic rather than a cytotoxic effect. A similar pattern of cytotoxicity was observed, for example, for antitubulin compounds in the nanomolar concentration range.
For another group of compounds (3f and 3k, etc.), there was a different pattern of cytotoxicity, when complete cell death was observed at high concentrations. Therefore, two parameters of action on the cells were calculated for the compounds: IC50 (half of the maximal inhibition) and IC50abs (half of the absolute quantity of cells) (see Table 3). The rest of the compounds had a low cytotoxicity or did not show noticeable toxic effects in the studied concentration range.

2.3. In Silico Characterization of Substituted Indoles Binding to Long RSH Proteins

Molecular Docking of Substituted Indoles

Searching for new chemical compounds that are targeted to non-essential bacterial adaptation pathways, such as stringent response, is an attractive approach for developing antimicrobials preventing bacterial persistence, biofilm formation, quorum sensing, and virulence [56]. Previously, researchers have identified indole derivatives as a chemotype capable of binding the RelSeq (p)ppGpp synthetase/hydrolase synthetic domain via virtual screening and thermal shift assays [26]. Based on these data, we selected a set of substituted indoles from our library of compounds to assess the possibility of their binding to RelMtb or RelSeq proteins in synthetic domain (SYN) active sites by using molecular docking methods. The RelMtb crystal structure unbound to the substrate [57] was used to analyze the ligand binding to an inactive state synthetase, while the RelSeq structure complexed with a GDP (guanosine diphosphate) substrate [58] was used to investigate the binding to (p)ppGpp synthetase in a catalytically competent state. The reference ligand indole-5-carboxylic acid demonstrated a RelSeq active site binding energy of −6.42 kcal/mol (Table 4), consistent with the result of −6.41 kcal/mol calculated in the previous research (in which ligand mentioned as I2) [26].
The docked ligands interacted with the Arg241 and His312 residues (Figure 4b,c), which are involved in GDP substrate binding [58]. Indolylquinazolinones and their derivatives demonstrated weaker binding in the RelSeq active site than the reference ligand, with the most negative docking score of −4.76 kcal/mol (Table 4). This might have been due to the planar structure of the analyzed compounds, lacking the flexibility to optimally fit the tight binding pocket in the substrate-bound state (Figure 4b,c). However, eight analyzed compounds demonstrated stronger binding energies (from −7.61 to −6.56 kcal/mol) compared to the reference ligand (−6.55 kcal/mol) in case of the substrate-unbound RelMtb structure SYN active site (Table 4).
Moreover, the indole derivatives interacted with the Tyr309 residue (Figure 5a–c), which has been shown to be essential for RelMtb (p)ppGpp synthesis [59]. Indole derivatives are therefore promising candidates for developing inhibitors of (p)ppGpp synthetases.

3. Materials and Methods

3.1. Instrumentation

1H and 13C NMR spectra were recorded on a «Bruker Avance III HD 400» (400 MHz for 1H and 100 MHz for 13C NMR) at 40 °C. The chemical shifts (δ) were measured in ppm with respect to the solvent ([D6] DMSO, 1H: δ = 2.50 ppm, 13C: δ = 39.52 ppm). The coupling constants (J) are given in Hertz (Hz). The splitting patterns of an apparent multiplets associated with the averaged coupling constants were designated as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), dd (doublet of doublets), ddd (doublet of doublet of doublets), and br. (broadened). High-resolution mass measurements (HRMS) were carried out using a BrukermicroTOF-QTM ESI-TOF mass spectrometer. A GC/MS analysis was performed on an «Agilent 7890B» interfaced to an «Agilent 5977A» mass selective detector. The melting points were determined with a «Stuart SMP 30». Data sets for X-Ray diffraction were collected with a «New Xcalibur, Ruby» diffractometer. Column chromatography was performed on silica gel Macherey Nagel (40–63 μm). Flash column chromatography was performed over silica gel (0.04–0.063 mm), using a mixture of EtOAc, petroleum ether. TLC plates were visualized by exposure to ultraviolet light. All the reactions were carried out using freshly distilled and dry solvents from solvent stills.

3.2. Materials

Starting 1H-indole-3-carboxaldehyde (purity 97%), furfural (99%), 5-methylfurfural (99%), 2-thiophenecarboxaldehyde (98%), 4-pyridinecarboxaldehyde (97%), 2-naphthaldehyde (98%), cyclohexanecarbaldehyde (97%), Lawesson’s reagent (97%), morpholine (99%), benzoyl chloride (99%), iodomethane (99%), and 2-aminobenzenesulfonamide (98%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The reagents for the analysis of the antimicrobial activity: Cefazolin—MP Biomedicals (Illkirch, France), Cefotaxime—PROMED (Saransk, Russia), Chloramphenicol—AppliChem (Darmstadt, Germany), Amikacin sulfate—Sigma-Aldrich (St. Louis, MO, USA), Fluconazole—Sigma-Aldrich (St. Louis, MO, USA), Amphotericin B—Sigma-Aldrich (St. Louis, MO, USA), Isoniazid—Sigma (St. Louis, MO, USA), LB-broth—VWR (Radnor, PA, USA), and LB-agar—Sigma (St. Louis, MO, USA).

3.3. Synthesis

3.3.1. Synthesis of the Starting Substrates

Starting 1-methylindole-3-carboxaldehyde (1b), 1-n-butyl-1H-indole-3-carbaldehyde (1e) [60], 1-ethyl-1H-indole-3-carbaldehyde (1c), 1-isopropyl-1H-indole-3-carbaldehyde (1d) [61], 1-benzyl-1H-indole-3-carbaldehyde (1f) [62], 1-phenyl-1H-indole-3-carbaldehyde (1g) [63], 1-acetyl-1H-indole-3-carbaldehyde (1h) [64], 1-benzoyl-1H-indole-3-carbaldehyde (1i) [65], 1-tosyl-1H-indole-3-carbaldehyde (1j) [66], 5-iodo-1H-indole-3-carbaldehyde (1k) [67], 5-bromo-1H-indole-3-carbaldehyde (1l) [68], 2-phenyl-1H-indole-3-carbaldehyde (1m) [69], 2-(4-nitrophenyl)-1H-indole-3-carbaldehyde (1n) [70], benzofuran-2-carbaldehyde (1p) [71], 5-ethylfuran-2-carbaldehyde (1s) [72], 1-methyl-1H-pyrrole-2-carbaldehyde (1t) [73], anthranilamide (2a) [74], 2-amino-4-chlorobenzamide (2b) [75], 2-aminobenzohydrazide (2e) [76], 2-hydroxybenzamide (2f) [77], 2-amino-5,6-dihydro-4H-cyclopentathiophene-3-carboxamide (2g) [78], and 2-amino-2-phenylacetamide (2i) [79] were synthesized according to known procedures.

3.3.2. Synthesis of 2-(1H-indol-3-yl)quinazolin-4(3H)-one (3a) [80]

To a solution of 1H-indole-3-carboxaldehyde (1a) (1.3 mmol, 0.189 g) and anthranilamide (2a) (1.3 mmol, 0.177 g) in dry acetonitrile (2.5 mL), p-TSA (0.5 mmol, 0.095 g) was added. The reaction mixture was refluxed for 4 h (TLC control). Then, the reaction mixture was poured into H2O (50 mL). The formed precipitate was filtered. The products were purified using column chromatography on silica gel using the mixture of petroleum ether/EtOAc (1:1) as an eluent and recrystallized from ethanol. There was a yield of 0.112 g (33%) and it was a colorless oil. 1H NMR (400 MHz, DMSO-d6) δ = 12.08 (br.s, 1H), 11.81 (br.s, 1H), 8.72—8.70 (m, 1H), 8.55 (d, J = 2.8 Hz, 1H), 8.13–8.11 (m, 1H), 7.81–7.72 (m, 2H), 7.50–7.48 (m, 1H), 7.43–7.39 (m, 1H), and 7.27–7.20 (m, 2H) ppm; 13C{1H} NMR (100 MHz, DMSO-d6) δ = 162.0, 150.2, 149.7, 136.8, 134.2, 129.0, 126.9, 125.7, 125.5, 125.0, 122.5, 122.3, 120.8, 120.4, 111.9, and 108.6 ppm.
Indole (4a) and quinazolin-4(3H)-one (5a) were isolated as by-products in the synthesis of 2-(1H-indol-3-yl)quinazolin-4(3H)-one (3a). The spectral data of the indole (4a) were identical to the commercially available ones. Quinazolin-4(3H)-one (5a) [81]. There was a yield of 0.023 g (12%) and it was a white solid. Mp. = 215–216 °C (EtOH, lit. [81] 213–214 °C). 1H NMR (400 MHz, DMSO-d6) δ = 12.16 (br.s, 1H), 8.12 (br.d, J = 8.0 Hz, 1H), 8.08 (s, 1H), 7.82–7.78 (m, 1H), 7.66 (d, J = 8.0 Hz, 1H), and 7.53–7.49 (m, 1H) ppm; 13C{1H} NMR (100 MHz, DMSO-d6) δ = 160.6, 148.7, 145.2, 134.2, 127.1, 126.6, 125.7, and 122.6 ppm.

3.3.3. Synthesis of 2-(1H-indol-3-yl)-2,3-dihydroquinazolin-4(1H)-one (6a) [82]

To a suspension of 1H-indole-3-carboxaldehyde (1a) (1.3 mmol, 0.189 g) and Al2O3 (acidic) (2.3 mmol, 0.235 g) in dry acetonitrile (2.5 mL), anthranilamide (2a) (1.3 mmol, 0.177 g) was added. The reaction mixture was refluxed for 55 h (TLC control). Then, the Al2O3 was filtered off and washed with hot EtOAc (3 × 5 mL). The combined organic fractions were concentrated to dryness under reduced pressure. The product was purified using column chromatography on silica gel using the mixture of petroleum ether/EtOAc (1:1) as an eluent. There was a yield of 0.154 g (45%) and it was a colorless oil. 1H NMR (400 MHz, DMSO-d6) δ = 11.09 (br.s, 1H), 8.08 (br.s, 1H), 7.80–7.78 (m, 1H), 7.70–7.68 (m, 1H), 7.43–7.41 (m, 2H), 7.27–7.23 (m, 1H), 7.15–7.11 (m, 1H), 7.04–7.01 (m, 1H), 6.92 (br.s, 1H), 6.79–6.77 (m, 1H), 6.73–6.69 (m, 1H), and 6.07 (s, 1H) ppm; 13C{1H} NMR (100 MHz, DMSO-d6) δ = 164.2, 148.8, 136.6, 133.0, 127.5, 125.4, 124.6, 121.4, 120.0, 118.7, 117.0, 115.3, 114.43, 114.38, 111.6, and 61.7 ppm.
Indole (4a) was isolated as by-product in the synthesis of 2-(1H-indol-3-yl)-2,3-dihydroquinazolin-4(1H)-one (6a). The spectral data of the indole (4a) were identical to the commercially available ones.

3.3.4. General Procedure for the Synthesis of Quinazolin-4(3H)-Ones 3a–3g, 3k–3ab

To a suspension of aldehyde 1 (1.3 mmol) and Na2S2O5 (4.5 mmol, 0.855 g) in N,N-dimethylacetamide (2.5 mL), amide 2 (1.3 mmol) and H2O (4.5 mmol, 81 μL) were added. The reaction mixture was heated at 150 °C for 6.5–55 h (TLC control). Then, the reaction mixture was poured into H2O (50 mL). The formed precipitate was filtered. The products 3a,d,f,g,k,l,o–r,t,w,x,ab were purified using column chromatography on silica gel using the mixture of petroleum ether/EtOAc (1:1) as an eluent and recrystallized from a suitable solvent. The products 3b,c,e,s,u,v,y,z,aa were recrystallized from a suitable solvent without prior purification using column chromatography.
2-(1H-Indol-3-yl)quinazolin-4(3H)-one (3a) [80]. A yield of 0.326 g (96%), 14 h, white solid. Mp. ≥ 320 °C, decomposition (EtOH, lit. [80] > 300 °C). All the spectral data of 2-(1H-indol-3-yl)quinazolin-4(3H)-one (3a) were consistent with those described above.
2-(1-Methyl-1H-indol-3-yl)quinazolin-4(3H)-one (3b) [83]. A yield of 0.211 g (59%), 43 h, white solid. Mp. ≥ 324 °C, decomposition (EtOH, lit. [83] 290–295 °C). 1H NMR (400 MHz, DMSO-d6) δ = 12.07 (s, 1H), 8.72–8.70 (m, 1H), 8.51 (s, 1H), 8.12–8.10 (m, 1H), 7.80–7.71 (m, 2H), 7.55 (br.d, J = 7.6 Hz, 1H), 7.43–7.39 (m, 1H), 7.33–7.25 (m, 2H), and 3.88 (s, 3H) ppm; 13C{1H} NMR (100 MHz, DMSO-d6) δ = 162.0, 149.9, 149.6, 137.3, 134.2, 132.8, 126.8, 125.9, 125.7, 125.0, 122.6, 122.4, 121.1, 120.4, 110.3, 107.6, and 33.2 ppm.
2-(1-Ethyl-1H-indol-3-yl)quinazolin-4(3H)-one (3c). A yield of 0.327 g (87%), 47 h, pale beige solid. Mp. = 283–284 °C (EtOH). 1H NMR (400 MHz, DMSO-d6) δ = 12.05 (br.s, 1H), 8.72 (d, J = 7.5 Hz, 1H), 8.62 (br.s, 1H), 8.12 (d, J = 7.6 Hz, 1H), 7.80–7.72 (m, 2H), 7.58 (d, J = 7.6 Hz, 1H), 7.43–7.39 (m, 1H), 7.31–7.24 (m, 2H), 4.27 (q, J = 7.2 Hz, 2H), and 1.48 (t, J = 7.2 Hz, 3H) ppm; 13C{1H} NMR (100 MHz, DMSO-d6) δ = 161.9, 149.9, 149.7, 136.5, 134.2, 131.1, 126.9, 126.0, 125.7, 125.0, 122.51, 122.50, 121.1, 120.4, 110.3, 107.7, 40.8, and 14.7 ppm. HRMS (ESI) Calcd for C18H16N3O [M + H]+ 290.1288; Found 290.1278.
2-(1-Isopropyl-1H-indol-3-yl)quinazolin-4(3H)-one (3d). A yield of 0.319 g (81%), 24 h, beige solid. Mp. = 275–276 °C (EtOH). 1H NMR (400 MHz, DMSO-d6) δ = 12.03 (br.s, 1H), 8.78 (br.s, 1H), 8.73–8.71 (m, 1H), 8.10 (br.d, J = 7.6 Hz, 1H), 7.80–7.72 (m, 2H), 7.62 (br.d, J = 7.6 Hz, 1H), 7.43–7.39 (m, 1H), 7.31–7.24 (m, 2H), 4.89–4.80 (m, 1H), 1.54 (s, 3H), and 1.52 (s, 3H) ppm; 13C{1H} NMR (100 MHz, DMSO-d6) δ = 161.9, 150.0, 149.7, 136.3, 134.2, 128.5, 126.9, 126.0, 125.7, 125.0, 122.5, 122.4, 121.1, 120.3, 110.4, 107.8, 47.1, and 22.3 (2C) ppm. HRMS (ESI) Calcd for C19H18N3O [M + H]+ 304.1444; Found 304.1452.
2-(1-Butyl-1H-indol-3-yl)quinazolin-4(3H)-one (3e). A yield of 0.317 g (77%), 55 h, white solid. Mp. = 264–265 °C (EtOH). 1H NMR (400 MHz, DMSO-d6) δ = 12.05 (br.s, 1H), 8.72 (br.d, J = 8.0 Hz, 1H), 8.59 (s, 1H), 8.12–8.10 (m, 1H), 7.80–7.71 (m, 2H), 7.60 (d, J = 8.0 Hz, 1H), 7.43–7.39 (m, 1H), 7.31–7.24 (m, 2H), 4.25 (t, J = 7.2 Hz, 2H), 1.88–1.81 (m, 2H), 1.36–1.31 (m, 2H), and 0.94 (t, J = 7.2 Hz, 3H) ppm; 13C{1H} NMR (100 MHz, DMSO-d6) δ = 161.9, 149.9, 149.6, 136.8, 134.2, 131.8, 126.8, 125.9, 125.6, 125.0, 122.51, 122.48, 121.0, 120.3, 110.4, 107.6, 45.6, 31.2, 19.3, and 13.4 ppm. HRMS (ESI) Calcd for C20H20N3O [M + H]+ 318.1601; Found 318.1598.
2-(1-Benzyl-1H-indol-3-yl)quinazolin-4(3H)-one (3f). A yield of 0.411 g (90%), 19 h, white solid. Mp. = 307–308 °C (EtOH). 1H NMR (400 MHz, DMSO-d6) δ = 12.12 (br.s, 1H), 8.73–8.71 (m, 1H), 8.66 (br.s, 1H), 8.11 (d, J = 7.6 Hz, 1H), 7.80–7.72 (m, 2H), 7.63–7.60 (m, 1H), 7.43–7.40 (m, 1H), 7.36–7.33 (m, 4H), 7.31–7.24 (m, 3H), and 5.50 (s, 2H) ppm; 13C{1H} NMR (100 MHz, DMSO-d6) δ = 161.9, 149.8, 149.6, 137.0, 136.7, 134.2, 132.2, 128.6 (2C), 127.6, 127.4 (2C), 126.9, 126.1, 125.7, 125.1, 122.7, 122.5, 121.2, 120.4, 110.7, 108.3, and 49.7 ppm. HRMS (ESI) Calcd for C23H18N3O [M + H]+ 352.1444; Found 352.1451.
2-(1-Phenyl-1H-indol-3-yl)quinazolin-4(3H)-one (3g). A yield of 0.377 g (86%), 14 h, pale yellow solid. Mp. = 344–345 °C (EtOH). 1H NMR (400 MHz, DMSO-d6) δ = 12.14 (br.s, 1H), 8.86 (s, 1H), 8.84–8.82 (m, 1H), 8.14 (br.d, J = 8.0 Hz, 1H), 7.84–7.78 (m, 2H), 7.71–7.67 (m, 4H), 7.65–7.61 (m, 1H), 7.53–7.50 (m, 1H), 7.48–7.45 (m, 1H), and 7.37–7.35 (m, 2H) ppm; 13C{1H} NMR (100 MHz, DMSO-d6) δ = 161.9, 138.9, 138.1, 136.1, 134.3, 131.7, 130.0 (2C), 127.4, 127.1, 126.6, 125.7, 125.5, 124.0 (2C), 123.7, 122.9, 122.0, 120.6, 110.8, 110.6, and 110.1 ppm. HRMS (ESI) Calcd for C22H16N3O [M + H]+ 338.1288; Found 338.1288.
2-(5-Iodo-1H-indol-3-yl)quinazolin-4(3H)-one (3k) [8]. A yield of 0.478 g (95%), 14.5 h, beige solid. Mp. ≥ 308 °C, decomposition (EtOH, lit. [8] 309–311 °C). 1H NMR (400 MHz, DMSO-d6) δ = 12.13 (br.s, 1H), 11.98 (br.s, 1H), 9.06 (d, J = 1.6 Hz, 1H), 8.53 (s, 1H), 8.12 (dd, J = 8.0, 0.8 Hz, 1H), 7.81–7.78 (m, 1H), 7.73–7.70 (m, 1H), 7.51 (dd, J = 8.4, 1.6 Hz, 1H), 7.45–7.41 (m, 1H), and 7.36 (d, J = 8.4 Hz, 1H) ppm; 13C{1H} NMR (100 MHz, DMSO-d6) δ = 161.9, 149.8, 149.4, 135.9, 134.3, 130.63, 130.56, 129.8, 127.9, 126.9, 125.7, 125.2, 120.4, 114.4, 107.8, and 85.1 ppm.
2-(5-Bromo-1H-indol-3-yl)quinazolin-4(3H)-one (3l). A yield of 0.331 g (75%), 14 h, beige solid. Mp. ≥ 340 °C, decomposition (EtOH). 1H NMR (400 MHz, DMSO-d6) δ = 12.14 (br.s, 1H), 12.01 (br.s, 1H), 8.85 (d, J = 1.6 Hz, 1H), 8.58 (s, 1H), 8.12 (dd, J = 8.0, 0.8 Hz, 1H), 7.81–7.72 (m, 2H), 7.47 (d, J = 8.4 Hz, 1H), 7.45–7.41 (m, 1H), and 7.37 (dd, J = 8.8, 2.0 Hz, 1H) ppm; 13C{1H} NMR (100 MHz, DMSO-d6) δ = 161.9, 149.8, 149.4, 135.5, 134.3, 130.2, 127.2, 126.9, 125.7, 125.2, 125.1, 124.4, 120.4, 114.0, 113.6, and 108.2 ppm. HRMS (ESI) Calcd for C16H11BrN3O [M + H]+ 340.0080; Found 340.0080.
2-(2-Phenyl-1H-indol-3-yl)quinazolin-4(3H)-one (3m) was determined using GC/MS in trace amounts. Quinazolin-4(3H)-one (5a) was isolated as by-product in the synthesis of 2-(2-phenyl-1H-indol-3-yl)quinazolin-4(3H)-one (3m) in a 37% yield. All the spectral data of quinazolin-4(3H)-one (5a) were consistent with those described above.
2-[2-(4-Nitrophenyl)-1H-indol-3-yl]quinazolin-4(3H)-one (3n) was determined using GC/MS in trace amounts. Quinazolin-4(3H)-one (5a) was isolated as by-product in the synthesis of 2-[2-(4-nitrophenyl)-1H-indol-3-yl]quinazolin-4(3H)-one (3n) in a 59% yield. All the spectral data of quinazolin-4(3H)-one (5a) were consistent with those described above.
6-Chloro-2-(1H-indol-3-yl)quinazolin-4(3H)-one (3o). A yield of 0.350 g (91%), 20 h, pale beige solid. Mp. ≥ 390 °C, decomposition (EtOH). 1H NMR (400 MHz, DMSO-d6) δ = 12.24 (br.s, 1H), 11.85 (br.s, 1H), 8.68 (br.d, J = 6.8 Hz, 1H), 8.55 (d, J = 2.0 Hz, 1H), 8.04 (s, 1H), 7.79–7.73 (m, 2H), 7.49 (br.d, J = 6.8 Hz, 1H), and 7.26–7.20 (m, 2H) ppm; 13C{1H} NMR (100 MHz, DMSO-d6) δ = 161.1, 150.7, 148.4, 136.8, 134.3, 129.4, 129.1, 129.0, 125.4, 124.7, 122.6, 122.3, 121.5, 120.9, 111.9, and 108.3 ppm. HRMS (ESI) Calcd for C16H11ClN3O [M + H]+ 296.0585; Found 296.0582.
7-Chloro-2-(1H-indol-3-yl)quinazolin-4(3H)-one (3p). A yield of 0.357 g (93%), 23 h, pale beige solid. Mp. = 323–324 °C (EtOH). 1H NMR (400 MHz, DMSO-d6) δ = 12.19 (br.s, 1H), 11.87 (br.s, 1H), 8.70 (d, J = 7.2 Hz, 1H), 8.57 (d, J = 2.4 Hz, 1H), 8.09 (d, J = 8.4 Hz, 1H), 7.78 (s, 1H), 7.50–7.48 (m, 1H), 7.42–7.40 (m, 1H), and 7.26–7.20 (m, 2H) ppm; 13C{1H} NMR (100 MHz, DMSO-d6) δ = 161.4, 151.6, 150.9, 138.9, 136.8, 129.6, 127.7, 125.9, 125.5, 125.1, 122.6, 122.4, 121.0, 119.1, 111.9, and 108.3 ppm. HRMS (ESI) Calcd for C16H11ClN3O [M + H]+ 296.0585; Found 296.0582.
2-(1H-Indol-3-yl)-6-iodoquinazolin-4(3H)-one (3q). A yield of 0.226 g (45%), 32 h, pale brown solid. Mp. ≥ 308 °C, decomposition (1,4-dioxane/EtOH). 1H NMR (400 MHz, DMSO-d6) δ = 12.21 (br.s, 1H), 11.85 (br.s, 1H), 8.68–8.66 (m, 1H), 8.54 (br.s, 1H), 8.37 (br.s, 1H), 8.05–8.03 (m, 1H), 7.54–7.48 (m, 2H), and 7.25–7.20 (m, 2H) ppm; 13C{1H} NMR (100 MHz, DMSO-d6) δ = 160.7, 150.8, 149.0, 142.5, 136.8, 134.0, 129.4, 129.1, 125.4, 122.6, 122.3, 122.2, 120.9, 111.9, 108.4, and 89.1 ppm. HRMS (ESI) Calcd for C16H11IN3O [M + H]+ 387.9941; Found 387.9947.
N,N-dimethyl-1H-indole-3-carbothioamide (7a) was isolated as a by-product in the synthesis of 2-(1H-indol-3-yl)-6-iodoquinazolin-4(3H)-one (3q), 3-(1H-indol-3-yl)-2H-benzo[e][1,2,4]thiadiazine-1,1-dioxide (3af), and 2-(1H-indol-3-yl)-5-phenyl-3,5-dihydro-4H-imidazol-4-one (3ag) in 21%, 47%, and 50% yields, respectively. Yields of 0.056 g (21%) for 3q, 0.125 g (47%) for 3af, and 0.133 g (50%) for 3ag were obtained and they were pale beige solids. Mp. ≥ 180 °C, decomposition (petroleum ether/EtOAc). 1H NMR (400 MHz, DMSO-d6) δ = 11.51 (br.s, 1H), 7.68–7.62 (m, 2H), 7.43 (s, 1H), 7.12 (br.s, 2H), and 3.42 (br.s, 6H) ppm; 13C{1H} NMR (100 MHz, DMSO-d6) δ = 193.2, 135.5, 127.0, 124.8, 121.6, 120.3, 119.9, 118.3, 111.8, and 43.3 (br.s, 2C) ppm. HRMS (ESI) Calcd for C11H13N2S [M + H]+ 205.0794; Found 205.0792. (CCDC 2269768 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via. Available online: www.ccdc.cam.ac.uk/structures; accessed on 14 June 2023; for details, see Supplementary Materials).
2-(1H-Indol-2-yl)quinazolin-4(3H)-one (3r) [84]. A yield of 0.271 g (80%), 15 h, grey solid. Mp. ≥ 326 °C, decomposition (EtOH, lit. [84] 302–304 °C). 1H NMR (400 MHz, DMSO-d6) δ = 12.54 (br.s, 1H), 11.73 (br.s, 1H), 8.16 (br.d, J = 7.6 Hz, 1H), 7.86–7.83 (m, 1H), 7.75–7.73 (m, 1H), 7.67–7.64 (m, 2H), 7.56–7.49 (m, 2H), 7.25–7.22 (m, 1H), and 7.09–7.05 (m, 1H) ppm; 13C{1H} NMR (100 MHz, DMSO-d6) δ = 161.6, 148.7, 146.5, 137.6, 134.5, 130.0, 127.4, 126.8, 126.1, 126.0, 123.9, 121.4, 121.1, 119.9, 112.3, and 104.9 ppm.
2-(Benzofuran-2-yl)quinazolin-4(3H)-one (3s) [85]. A yield of 0.235 g (69%), 6,5 h, beige solid. Mp. = 253–254 °C (EtOH) (lit. [85] 254–257 °C). 1H NMR (400 MHz, DMSO-d6) δ = 12.69 (br.s, 1H), 8.17 (br.d, J = 7.6 Hz, 1H), 8.06 (s, 1H), 7.85–7.73 (m, 4H), 7.57–7.47 (m, 2H), and 7.37–7.34 (m, 1H) ppm; 13C{1H} NMR (100 MHz, DMSO-d6) δ = 161.4, 154.8, 148.3, 147.6, 144.2, 134.6, 127.4, 127.3, 126.93, 126.91, 125.9, 123.7, 122.5, 121.5, 111.7, and 110.1 ppm.
2-(Furan-2-yl)quinazolin-4(3H)-one (3t) [84]. A yield of 0.234 g (85%), 9 h, beige solid. Mp. = 221–222 °C (EtOH) (lit. [84] 210–212 °C). 1H NMR (400 MHz, DMSO-d6) δ = 12.42 (br.s, 1H), 8.12 (br.d, J = 7.2 Hz, 1H), 7.98 (br.s, 1H), 7.82–7.78 (m, 1H), 7.69–7.67 (m, 1H), 7.62 (d, J = 3.2 Hz, 1H), 7.50–7.47 (m, 1H), and 6.74–6.73 (m, 1H) ppm; 13C{1H} NMR (100 MHz, DMSO-d6) δ = 161.4, 148.6, 146.4, 146.1, 143.9, 134.5, 127.1, 126.3, 125.8, 121.1, 114.3, and 112.4 ppm.
2-(5-Methylfuran-2-yl)quinazolin-4(3H)-one (3u) [86]. A yield of 0.209 g (71%), 9 h, brown solid. Mp. = 195–196 °C (EtOH) (lit. [86] 191–192 °C). 1H NMR (400 MHz, DMSO-d6) δ = 12.28 (br.s, 1H), 8.11 (d, J = 7.6 Hz, 1H), 7.81–7.77 (m, 1H), 7.69–7.67 (m, 1H), 7.53 (d, J = 3.0 Hz, 1H), 7.48–7.44 (m, 1H), 6.36 (d, J = 3.0 Hz, 1H), and 2.41 (s, 3H) ppm; 13C{1H} NMR (100 MHz, DMSO-d6) δ = 161.4, 156.0, 148.8, 144.4, 143.9, 134.4, 127.0, 126.0, 125.8, 120.9, 115.7, 108.8, and 13.4 ppm.
2-(5-Ethylfuran-2-yl)quinazolin-4(3H)-one (3v). A yield of 0.265 g (85%), 9 h, pale yellow solid. Mp. = 187–188 °C (EtOH). 1H NMR (400 MHz, DMSO-d6) δ = 12.30 (br.s, 1H), 8.11 (d, J = 8.0 Hz, 1H), 7.80–7.77 (m, 1H), 7.69–7.67 (m, 1H), 7.52 (d, J = 3.0 Hz, 1H), 7.48–7.44 (m, 1H), 6.37 (d, J = 3.0 Hz, 1H), 2.75 (q, J = 7.6 Hz, 2H), and 1.25 (t, J = 7.6 Hz, 3H) ppm; 13C{1H} NMR (100 MHz, DMSO-d6) δ = 161.5, 161.3, 148.8, 144.3, 144.0, 134.4, 127.0, 126.0, 125.8, 120.9, 115.5, 107.4, 20.9, and 11.7 ppm. HRMS (ESI) Calcd for C14H13N2O2 [M + H]+ 241.0972; Found 241.0971.
2-(1-Methyl-1H-pyrrol-2-yl)quinazolin-4(3H)-one (3w). A yield of 0.219 g (75%), 10 h, white solid. Mp. = 224 °C (EtOH). 1H NMR (400 MHz, DMSO-d6) δ = 11.98 (br.s, 1H), 8.09 (d, J = 7.6 Hz, 1H), 7.78–7.74 (m, 1H), 7.63–7.61 (m, 1H), 7.44–7.40 (m, 1H), 7.22 (d, J = 2.0 Hz, 1H), 7.08 (s, 1H), 6.15 (br.s, 1H), and 4.07 (s, 3H) ppm; 13C{1H} NMR (100 MHz, DMSO-d6) δ = 161.8, 148.7, 146.7, 134.2, 129.9, 126.9, 125.6, 125.5, 123.9, 120.3, 114.7, 107.6, and 37.4 ppm. HRMS (ESI) Calcd for C13H12N3O [M + H]+ 226.0975; Found 226.0979.
2-(Thiophen-2-yl)quinazolin-4(3H)-one (3x) [84]. A yield of 0.291 g (98%), 14.5 h, pale yellow solid. Mp. = 287 °C (EtOH) (lit. [84] 246–248 °C). 1H NMR (400 MHz, DMSO-d6) δ = 12.58 (br.s, 1H), 8.23 (d, J = 2.8 Hz, 1H), 8.13–8.12 (m, 1H), 7.85–7.78 (m, 2H), 7.66–7.64 (m, 1H), 7.50–7.46 (m, 1H), and 7.24–7.22 (m, 1H) ppm; 13C{1H} NMR (100 MHz, DMSO-d6) δ = 161.7, 148.5, 147.8, 137.3, 134.5, 132.0, 129.3, 128.3, 126.8, 126.2, 125.9, and 120.8 ppm.
2-(Pyridin-4-yl)quinazolin-4(3H)-one (3y) [84]. A yield of 0.044 g (15%), 7.5 h, white solid. Mp. = 276–277 °C (EtOH) (lit. [84] 270–272 °C). 1H NMR (400 MHz, DMSO-d6) δ = 12.69 (br.s, 1H), 8.78 (d, J = 4.4 Hz, 2H), 8.18 (br.d, J = 7.6 Hz, 1H), 8.11 (d, J = 4.4 Hz, 2H), 7.88–7.84 (m, 1H), 7.79–7.77 (m, 1H), and 7.59–7.55 (m, 1H) ppm; 13C{1H} NMR (100 MHz, DMSO-d6) δ = 161.9, 150.4, 150.1 (2C), 148.2, 139.8, 134.6, 127.6, 127.2, 125.8, 121.4 (2C), and 121.4 ppm.
2-(Naphthalen-2-yl)quinazolin-4(3H)-one (3z) [83]. A yield of 0.301 g (85%), 9 h, pale yellow solid. Mp. = 285–286 °C (EtOH) (lit. [83] 285–287 °C). 1H NMR (400 MHz, DMSO-d6) δ = 12.59 (br.s, 1H), 8.82 (br.s, 1H), 8.32–8.30 (m, 1H), 8.19 (d, J = 7.6 Hz, 1H), 8.08–8.06 (m, 2H), 8.02–8.00 (m, 1H), 7.88–7.84 (m, 1H), 7.81–7.79 (m, 1H), 7.66–7.61 (m, 2H), and 7.56–7.52 (m, 1H) ppm; 13C{1H} NMR (100 MHz, DMSO-d6) δ = 162.2, 152.2, 148.6, 134.5, 134.1, 132.2, 129.9, 128.8, 128.03, 127.97, 127.8, 127.5, 127.4, 126.8, 126.5, 125.8, 124.4, and 121.0 ppm.
2-Cyclohexylquinazolin-4(3H)-one (3aa) [87]. A yield of 0.261 g (88%), 8 h, white solid. Mp. = 222 °C (EtOAc) (lit. [87] 230–231 °C). 1H NMR (400 MHz, DMSO-d6) δ = 11.99 (br.s, 1H), 8.07 (br.d, J = 7.6 Hz, 1H), 7.77–7.73 (m, 1H), 7.58 (br.d, J = 8.0 Hz, 1H), 7.45–7.42 (m, 1H), 2.61–2.55 (m, 1H), 1.92–1.89 (m, 2H), 1.80–1.77 (m, 2H), 1.69–1.55 (m, 3H), and 1.35–1.20 (m, 3H) ppm; 13C{1H} NMR (100 MHz, DMSO-d6) δ = 161.8, 160.6, 148.9, 134.0, 126.9, 125.7, 125.5, 120.9, 42.7, 30.1 (2C), 25.4 (2C), and 25.2 ppm.
3-Amino-2-(1H-indol-3-yl)quinazolin-4(3H)-one (3ab). A yield of 0.212 g (59%), 15 h, pale brown solid. Mp. ≥ 235 °C, decomposition (EtOH). 1H NMR (400 MHz, DMSO-d6) δ = 11.78 (br.s, 1H), 8.78–8.75 (m, 2H), 8.15–8.13 (m, 1H), 7.82–7.74 (m, 2H), 7.53–7.50 (m, 1H), 7.46–7.43 (m, 1H), 7.25–7.20 (m, 2H), and 5.97 (s, 2H) ppm; 13C{1H} NMR (100 MHz, DMSO-d6) δ = 161.0, 151.0, 147.4, 135.8, 133.8, 133.0, 127.0, 126.7, 125.8, 125.0, 122.8, 122.1, 120.6, 118.4, 111.6, and 107.7 ppm. HRMS (ESI) Calcd for C16H13N4O [M + H]+ 277.1084; Found 277.1082.

3.3.5. Synthesis of 2-(5-methylfuran-2-yl)-2,3-dihydroquinazolin-4(1H)-one (6u) [88]

To a solution of 5-methylfuran-2-carbaldehyde (1s) (0.76 mmol, 76 µL) in DMSO (1.0 mL), anthranilamide (2a) (0.69 mmol, 94 mg) was added. The reaction mixture was heated at 100 °C for 3.5 h (TLC control). Then, the reaction mixture was poured into H2O (50 mL). The formed precipitate was filtered. The product was purified using column chromatography on silica gel using the mixture of petroleum ether/EtOAc (1:1) as an eluent and recrystallized from ethanol. A yield of 0.098 g (62%) was obtained and it was a pale yellow solid. Mp. = 168–169 °C (EtOH) (lit. [88] 177–178 °C). 1H NMR (400 MHz, DMSO-d6) δ = 8.24 (br.s, 1H), 7.61 (br.d, J = 7.6 Hz, 1H), 7.25–7.21 (m, 1H), 7.10 (s, 1H), 6.75 (br.d, J = 8.4 Hz, 1H), 6.69–6.65 (m, 1H), 6.13 (d, J = 3.2 Hz, 1H), 5.98 (br.s, 1H), 5.68 (br.s, 1H), and 2.22 (s, 3H) ppm; 13C{1H} NMR (101 MHz, DMSO-d6) δ = 163.1, 152.5, 151.3, 147.1, 133.1, 127.1, 117.1, 114.9, 114.4, 108.0, 106.2, 60.3, and 13.2 ppm.

3.3.6. Synthesis of 2-(thiophen-2-yl)-2,3-dihydro-4H-benzo[e][1,3]oxazin-4-one (3ad) [89]

To a solution of thiophene-2-carbaldehyde (1u) (0.25 mmol, 24 μL) in toluene (2 mL), 2-hydroxybenzamide (1f) (0.25 mmol, 0.034 g) and TFA (0.25 mmol, 19 μL) were added. The reaction mixture was refluxed for 40 h (TLC control) and cooled to room temperature. The reaction mixture was poured into H2O (10 mL) and extracted with EtOAc (3 × 5 mL). The combined organic layers were washed with brine (3 × 10 mL), dried with anhydrous Na2SO4, and concentrated to dryness. The product was purified using column chromatography on silica gel using the mixture of petroleum ether/EtOAc (5:1) as an eluent. A yield of 0.030 g (52%) was obtained and it was a yellow oil. 1H NMR (400 MHz, DMSO-d6) δ = 9.14 (br.s, 1H), 7.79 (br.d, J = 7.6 Hz, 1H), 7.62–7.61 (m, 1H), 7.53–7.49 (m, 1H), 7.28–7.27 (m, 1H), 7.14–7.11 (m, 1H), 7.05–7.03 (m, 2H), and 6.69 (br.s, 1H) ppm; 13C{1H} NMR (101 MHz, DMSO-d6) δ = 162.0, 156.1, 140.7, 134.5, 127.8, 127.7, 127.3, 126.7, 122.4, 118.3, 116.9, and 80.5 ppm.

3.3.7. Synthesis of 2-(1-benzoyl-1H-indol-3-yl)quinazolin-4(3H)-one (3i)

To a solution of 2-(1H-indol-3-yl)quinazolin-4(3H)-one (3a) (1.0 mmol, 0.261 g) and triethylamine (2.0 mmol, 279 µL) in dimethylacetamide (2.0 mL) benzoyl chloride (2.0 mmol, 232 µL) was added. The reaction mixture was heated at 150 °C for 4 h (TLC control). Then, the reaction mixture was poured into H2O (50 mL). Then, NaHCO3 (4.2 mmol, 353 mg) was added portion wise. The reaction mixture was stirred at room temperature for 2 h. The formed precipitate was filtered. The product 3i was purified using column chromatography on silica gel using the mixture of petroleum ether/EtOAc (2:1) as an eluent and recrystallized from an acetone. A yield of 0.288 g (79%) was obtained and it was a white solid. Mp. = 317–318 °C, decomposition (acetone). 1H NMR (400 MHz, DMSO-d6) δ = 12.45 (br.s, 1H), 8.89–8.87 (m, 1H), 8.76 (s, 1H), 8.38–8.36 (m, 1H), 8.14–8.12 (m, 1H), 7.88–7.82 (m, 4H), 7.79–7.75 (m, 1H), 7.70–7.66 (m, 2H), and 7.52–7.48 (m, 3H) ppm; 13C{1H} NMR (100 MHz, DMSO-d6) δ = 168.4, 161.8, 148.8, 148.5, 136.1, 134.4, 133.1, 132.4, 130.9, 129.5 (2C), 128.7 (2C), 127.8, 127.3, 126.3, 125.7, 125.5, 124.6, 123.1, 121.0, 115.6, and 113.9 ppm. HRMS (ESI) Calcd for C23H16N3O2 [M + H]+ 366.1237; Found 366.1246.

3.3.8. Synthesis of Quinazolin-4(3H)-Ones 3b, 3ah, 3ai

To a stirred solution of 2-(1H-indole-3-yl)quinazolin-4(3H)-one (3a) (0.9 mmol, 0.235 g) in dry DMF (1 mL), sodium hydride (2.7 mmol, 60% dispersion in mineral oil, 0.108 g) was added portion wise. The reaction mixture was stirred at room temperature for 5 min, followed by the addition of a methyl iodide (1.8 mmol, 112 µL). The reaction mixture was stirred for 48 h at ambient temperature (TLC control). The reaction mixture was poured into H2O (30 mL). The formed precipitate was filtered. The products were purified using column chromatography on silica gel using the mixture of petroleum ether/EtOAc (3:1) as an eluent and recrystallized from a suitable solvent.
2-(1-Methyl-1H-indol-3-yl)quinazolin-4(3H)-one (3b) [83]. A yield of 0.067 g (27%), white solid. Mp. ≥ 324 °C, decomposition (EtOH, lit. [83] 290–295 °C). All the spectral data of 2-(1-methyl-1H-indol-3-yl)quinazolin-4(3H)-one (3b) were consistent with those described above.
3-Methyl-2-(1-methyl-1H-indol-2-yl)quinazolin-4(3H)-one (3ah) [90]. A yield of 0.057 g (22%), white solid. Mp. = 192–193 °C (EtOH, lit. [90] 194–196 °C). 1H NMR (400 MHz, DMSO-d6) δ = 8.16 (dd, J = 8.0, 1.2 Hz, 1H), 8.06–8.04 (m, 2H), 7.82–7.78 (m, 1H), 7.66 (d, J = 8.0 Hz, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.50–7.46 (m, 1H), 7.32–7.28 (m, 1H), 7.23–7.20 (m, 1H), 3.92 (s, 3H), and 3.69 (s, 3H) ppm; 13C{1H} NMR (100 MHz, DMSO-d6) δ = 162.1, 151.8, 147.6, 136.3, 134.0, 132.7, 126.7, 126.6, 126.0, 125.7, 122.2, 121.2, 120.6, 119.2, 110.2, 108.7, 33.6, and 32.8 ppm.
4-Methoxy-2-(1-methyl-1H-indol-3-yl)quinazoline (3ai). A yield of 0.052 g (20%), yellow solid. Mp. = 160 °C, decomposition (EtOAc). 1H NMR (400 MHz, DMSO-d6) δ = 8.76–8.74 (m, 1H), 8.36 (s, 1H), 8.09–8.06 (m, 1H), 7.89–7.84 (m, 2H), 7.55–7.48 (m, 2H), 7.30–7.24 (m, 2H), 4.27 (s, 3H), and 3.92 (s, 3H) ppm; 13C{1H} NMR (100 MHz, DMSO-d6) δ = 165.7, 158.7, 151.4, 137.7, 134.0, 133.7, 126.7, 126.1, 125.2, 123.0, 122.2, 121.9, 120.7, 113.9, 113.7, 110.2, 53.9, and 32.8 ppm. HRMS (ESI) Calcd for C18H16N3O [M + H]+ 290.1288; Found 290.1279.

3.3.9. Synthesis of 2-(1H-indol-3-yl)-4-(methylthio)quinazoline (3ak)

To a solution of 2-(1H-indol-3-yl)quinazolin-4(3H)-one (3a) (0.5 mmol, 131 mg) in toluene (5 mL), Lawesson’s reagent (1.5 mmol, 606 mg) was added. The reaction mixture was refluxed for 4 h (TLC control) and cooled to room temperature. The mixture was treated with 1N sodium hydroxide (5 mL) and extracted with EtOAc (3 × 10 mL). The combined organic layers were washed with brine (3 × 10 mL), dried with anhydrous Na2SO4, and concentrated to dryness. To a solution of 2-(1H-indol-3-yl)quinazoline-4(3H)-thione (3aj) in acetone (2 mL), K2CO3 (0.6 mmol, 0.083 g) and MeI (0.6 mmol, 37 μL) were added. The reaction mixture was stirred for 2 h at room temperature (TLC control). The reaction was poured into H2O (100 mL) and extracted with EtOAc (3 × 20 mL). To the reaction, H2O (2 mL) was added and extracted with EtOAc (3 × 20 mL). The combined organic layers were washed with brine (3 × 10 mL), dried with anhydrous Na2SO4, and concentrated to dryness. The product was purified using column chromatography on silica gel using the mixture of petroleum ether/EtOAc (19:1) as an eluent and recrystallized from a mixture of petroleum ether/EtOAc. A yield of 0.076 g (52%), yellow solid. Mp. = 182–184 °C (petroleum ether/EtOAc). 1H NMR (400 MHz, DMSO-d6) δ = 11.67 (br.s, 1H), 8.76–8.73 (m, 1H), 8.40 (d, J = 2.8 Hz, 1H), 8.02 (d, J = 8.4 Hz, 1H), 7.93–7.86 (m, 2H), 7.55–7.50 (m, 2H), 7.23–7.21 (m, 2H), and 2.84 (s, 3H) ppm; 13C{1H} NMR (100 MHz, DMSO-d6) δ = 169.6, 158.3, 148.4, 137.2, 133.9, 130.3, 127.8, 125.7, 125.6, 123.4, 122.0, 121.9, 120.9, 120.4, 114.9, 111.9, and 12.0 ppm. HRMS (ESI) Calcd for C17H14N3S [M + H]+ 292.0903; Found 292.0894.

3.3.10. Synthesis of 4-Chloro-2-(1H-indol-3-yl)quinazoline (3am)

To a solution of 2-(1H-indol-3-yl)quinazolin-4(3H)-one (3a) (1.0 mmol, 0.261 g) in dry DMF (4.5 mL), POCl3 (9.0 mmol) was added at 0 °C. The reaction was stirred at ambient temperature for 12 h (TLC control). Then, the reaction mixture was poured into H2O (100 mL). Then, NaHCO3 (10.0 mmol, 0.840 g) was added portion wise. The reaction mixture was stirred at room temperature for 2 h. The formed precipitate was filtered. The product was purified using column chromatography on silica gel using the mixture of petroleum ether/EtOAc (2:1) as an eluent and recrystallized from a mixture of petroleum ether/EtOAc. A yield of 0.128 g (46%), pale pink solid. Mp. = 217 °C (petroleum ether/EtOAc). 1H NMR (400 MHz, DMSO-d6) δ = 9.54 (br.s, 1H), 8.77–8.74 (m, 2H), 8.29–8.21 (m, 2H), 8.13–8.06 (m, 2H), 7.80–7.77 (m, 1H), and 7.51–7.45 (m, 2H) ppm; 13C{1H} NMR (100 MHz, DMSO-d6) δ = 161.1, 156.6, 150.9, 135.7 (2C), 128.7 (2C), 128.1 (2C), 127.6, 125.5 (2C), 125.3, 124.8, 122.7, and 121.3 ppm. HRMS (ESI) Calcd for C16H11ClN3 [M + H]+ 280.0636; Found 280.0635.

3.3.11. Synthesis of 4-[2-(1H-indol-3-yl)quinazolin-4-yl]morpholine (3an)

To a solution of 4-chloro-2-(1H-indol-3-yl)quinazoline (3am) (1.0 mmol, 279 mg) and Et3N (1.5 mmol, 202 µL) in MeCN (5 mL), morpholine (1.2 mmol, 103 µL) was added. The reaction mixture was refluxed for 1.5 h (TLC control). Then, the reaction mixture was poured into H2O (50 mL) and the product was extracted with dichloromethane (3 × 20 mL). The combined organic layers were washed with brine (3 × 20 mL), dried with anhydrous Na2SO4, and concentrated to dryness under reduced pressure. The product was purified using column chromatography on silica gel using the mixture of petroleum ether/EtOAc (10:1) as an eluent. A yield of 0.261 g (79%), beige oil. 1H NMR (400 MHz, DMSO-d6) δ = 11.58 (br.s, 1H), 8.72–8.68 (m, 1H), 8.26 (d, J = 2.8 Hz, 1H), 7.97 (br.d, J = 7.6 Hz, 1H), 7.84 (dd, J = 8.4, 0.8 Hz, 1H), 7.78–7.74 (m, 1H), 7.50–7.46 (m, 1H), 7.44–7.39 (m, 1H), 7.21–7.16 (m, 2H), 3.89–3.87 (m, 4H), and 3.77–3.75 (m, 4H) ppm; 13C{1H} NMR (100 MHz, DMSO-d6) δ = 164.0, 158.4, 152.2, 137.1, 132.5, 129.5, 127.6, 125.8, 125.0, 123.9, 122.1, 121.6, 120.2, 115.3, 114.1, 111.8, 66.0 (2C), and 50.0 (2C) ppm. HRMS (ESI) Calcd for C20H19N4O [M + H]+ 331.1553; Found 331.1559.

3.4. Antibacterial Activity

The synthesized compounds were tested for their in vitro growth inhibitory and bactericidal (fungicidal) activity against Staphylococcus aureus (ATCC 25923), Staphylococcus aureus (MRSA, ATCC 43300), Staphylococcus epidermidis (ATCC 12228), Escherichia coli (ATCC 8739), Escherichia coli (ATCC 25922), Mycobacterium smegmatis (mc(2)155/ ATCC 700084), and Candida albicans (ATCC 10231). Cefazolin, cefotaxime, chloramphenicol, amikacin, rifampicin, isoniazid, fluconazole, and Amphotericin B were used as control antimicrobial (fungicidal) agents. The cells were grown overnight at 37 °C in a glass tube with 5 mL of LB-broth containing 1% of glucose for C. albicans and 0.05% Tween 80 for M. smegmatis. The grown cultures were then diluted 1:100 with fresh medium and cultivated for 5 h. The M. smegmatis culture was grown for 20 h with agitation on a shaker GFL1092 (GFL, Germany) (200 rpm, 37 °C). Then, the cultures were adjusted to OD 0.1 (A625) and diluted to 1:10 for M. smegmatis and 1:100 for the other microorganisms. The resulting suspension was used to determine the MIC using the serial dilutions method in 96-well plates with modifications. The synthesized compounds were dissolved in DMSO at a concentration of 20 mg/mL and a series of two-fold dilutions was prepared in the same solvent. Then, 10 μL of the solutions was added to the wells of the plate containing 190 μL of the cell suspension. To the control wells, 10 µL of DMSO was added. The plates were incubated at 37 °C under static conditions for 24 h (72 h for M. smegmatis) for the minimum inhibitory concentration determination. The minimum bactericidal concentration and MFC were determined as the lowest concentration of an antimicrobial agent required to achieve a 99.9% reduction in the initial inoculum. In total, 10 µL from each well of a plate for the MIC determination was inoculated on Petri dish with LB-agar (containing 1% of glucose for C. albicans), and colonies were checked for after incubation (24 h, 37 °C) to determine the MBC or MFC. The antitubercular activity of the synthesized compounds was studied with the M. tuberculosis H37Rv strain using the standard BASTEC MGIT 960 growth system (Becton Dickinson) [50]; 10 mg of the test substances was dissolved in 1 mL of DMSO, then mixed with Middlebrook 7H9 medium in a ratio of 1:9 and added to MGIT tubes, obtaining a series of two-fold dilutions with final concentrations from 1 to 100 μg/mL. The tubes initially contained 7 mL of Middlebrook Broth 7H9, 0.8 mL of MGIT OADC supplement, and oxygen-quenched fluorochrome, which changes the fluorescence as microorganisms grow. A total of 0.5 mL of a suspension of M. tuberculosis H37Rv in physiological saline containing 5 × 107 cells/mL was added to each tube. All the tubes were incubated at 37 °C for 41 days and examined for fluorescence daily.

3.5. Biofilm Assay

Biofilm formation was measured using the classical crystal violet test in microtiter plates [91]. The cells were grown overnight at 37 °C in a glass tube with 5 mL of LB-broth. The grown culture was then adjusted to OD 0.1 (A625) and added to the wells of the plates at 200 μL and cultivated for 24 h at 37 °C under static conditions. After that, the wells were washed twice with distilled water, stained for 30 min with 0.1% crystal violet, and washed three times. The dye was extracted with 95% ethanol over 30 min and the optical density (A570) was measured with microplate reader Tecan Infinite M200Pro (Tecan, Austria).

3.6. Cytotoxicity Activity

3.6.1. Cell Lines and Culture Conditions

The human cell lines MCF7′, A549, VA13, and HEK293T were maintained in DMEM/F-12 media containing 10% FBS, 50u/mL of penicillin, and 0.05 mg/mL of streptomycin (all products were from Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C in 5% CO2. The cell cultures were genotyped using STR and tested for the absence of mycoplasma.

3.6.2. Cytotoxicity Measurements (Mosmann Assay)

The measurements were carried out using the Mosmann assay (MTT) [92]; 2500 cells per well for the A549, HEK293T, and MCF7′ cell lines or 4000 cells per well for the VA13 cell line were plated out in 135 µL of DMEM-F12 (Gibco) media in a 96-well plate. The cells were incubated in a 5% CO2 incubator for the first 16 h without being treated. Then, 15 µL of media-DMSO solutions or suspensions of the tested substances were added to the cells (final DMSO concentrations in the media were 1% or less) and treated cells at 72 h (triplicate each). The MTT reagent then was added to the cells up to a final concentration of 0.5 g/L (10× stock solution in PBS was used) and incubated for 3 h at 37 °C in the incubator, under an atmosphere of 5% CO2. The MTT solution was then discarded and 140 µL of DMSO was added. The plates were swayed on a shaker (80 rpm) to solubilize the formazan. The absorbance was measured using a microplate reader (VICTOR X5 Plate Reader) at a wavelength of 555 nm to measure the formazan concentration and corresponding quantity of cells. The results were used to construct a dose–response graph and estimate the CC50 value using the Prism software (GraphPad Software, Boston, MA, USA). Both the IC50 (half of the maximal inhibition) and IC50abs (half of the absolute quantity of cells) were calculated.

3.7. Molecular Docking

Molecular docking was carried out using the Schrödinger Maestro software [93]. Our analysis was based on the protein structures of RelMtb from M. tuberculosis (PDB 5XNX) and RelSeq from Streptococcus dysgalactiae subsp. equisimilis (PDB 1VJ7), which were optimized and minimized with Protein Preparation Wizard and OPLS3 force field. Hydrogen atoms and disulfide bonds were added and water molecules were removed. LigPrep was used to prepare the ligands: ionization states (in pH range of 7 ± 2) and tautomers of the molecules were generated and the chirality of the molecules was determined based on their three-dimensional structure. Only the strongest energy binding isomersation state or tautomer is demonstrated in results for each compound. The RelMtb and RelSeq protein structures were aligned and the 15 Å cubic binding region was centered relative to the crystallized GDP substrate. Receptor grids were generated for both proteins. The standard precision (SP) docking method was applied with default parameters.

4. Conclusions

In summary, a series of 2-(1H-indol-3-yl)quinazolin-4(3H)-one 3 and analogues was synthesized. The analysis of their biological activity showed that some target compounds have antibacterial properties, ranging from good to outstanding. Thus, compound 3k showed an activity comparable to that of standard antimicrobial agents against C. albicans, S. aureus, and S. epidermidis and a high activity against MRSA. This compound also showed activity against M. tuberculosis. In addition, the ability of 3k to prevent the formation of bacterial biofilms in sublethal concentrations was found. The evaluation of their cytotoxic activity showed that some compounds, 3c, 3f, 3g, 3k, 3r, and 3z, exhibited significant antiproliferative activity against all the cancer cell lines studied. As a result of the study of in silico compounds, it was shown that the set of synthesized indole derivatives contained compounds with high enough negative values of the binding energy (−∆G) in both protein models used for the molecular docking, which demonstrates their potential ability for the high affinity binding of alarmone synthetases nearest to their active centers. This opens up the possibility of detecting representatives of substituted indoles that can demonstrate an ability to suppress persister cell formation through the inhibition of alarmone synthetases that are synthesized during the transition to the stationary phase.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules28145348/s1, Table S1: Antimicrobial data (MIC and MBC/MFC, µg/mL) for the quinazolinone derivatives 3, 5, 6, 7; Scheme S1: Synthesis of N,N-dimethyl-1H-indole-3-carbothioamides 7a, b; copies of 1H, 13C NMR spectra of target compounds; copies of HRMS of new compounds; X-ray crystallography data, Table S2: Experimental details for N,N-dimethyl-1H-indole-3-carbothioamide (7a, CCDC 2269768); Figure S1. Structure of the N,N-dimethyl-1H-indole-3-carbothioamide (7a, CCDC 2269768) according to the X-ray diffraction data; non-hydrogen atoms are shown as thermal vibration ellipsoids with a probability of 50%.

Author Contributions

Conceptualization, E.Y.M. and M.G.U.; methodology, E.Y.M.; software, A.G.T. and R.Y.S.; investigation, E.Y.M., E.R.N., R.O.S., L.Y.N., M.A.S. and D.A.S.; resources, E.Y.M.; writing, E.Y.M., M.G.U., A.G.T., R.Y.S., L.Y.N. and D.A.S.; supervision, E.Y.M.; project administration, E.Y.M.; funding acquisition, E.Y.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Russian Science Foundation (Grant number 22-23-00503, https://rscf.ru/project/22-23-00503/, accessed on 11 July 2023).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The supporting information includes NMR spectral and HRMS charts.

Acknowledgments

We thank Maksim Dmitriev (Perm State University) for the preparation of single-crystal X-ray data.

Conflicts of Interest

The authors declare no conflict of interest.

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Molecules 28 05348 g001 550
Figure 1. Selected examples of biological active quinazolinone derivatives.
Figure 1. Selected examples of biological active quinazolinone derivatives.
Molecules 28 05348 g001
Molecules 28 05348 sch001 550
Scheme 1. The key results of optimization of conditions for the model cyclocondensation reaction.
Scheme 1. The key results of optimization of conditions for the model cyclocondensation reaction.
Molecules 28 05348 sch001
Molecules 28 05348 sch002 550
Scheme 2. The synthesis of substituted indolylquinazolinones 3. a All reactions were performed at 1.3 mmol scale of aldehyde 1 and 1.3 mmol of amide 2. Isolated yields. b The reaction was performed at 3 mmol of aldehyde 1 and 3 mmol of amide 2. Isolated yield. c The 2-(1H-indol-3-yl)quinazolin-4(3H)-one (3a) was isolated in 80–96% yield (n.d.—not detected). d The formation of indolylquinazolinones 3m, n were detected using thin-layer chromatography (TLC) and gas chromatography–mass spectrometry (GC/MS). The quinazolinone 5a was isolated in 37% and 59% yields. e The formation of indole (4a) was determined using TLC and GC/MS. f The corresponding N,N-dimethyl-1H-indole-3-carbothioamide (7a) was also isolated in a 21–50% yield. g The reaction conditions: aldehyde 1 (0.76 mmol), amide 2 (0.69 mmol), DMSO (1 mL), 100 °C, 3.5 h. Isolated yield. h The reaction conditions: aldehyde 1 (0.25 mmol), amide 2 (0.25 mmol), TFA (0.25 mmol), toluene (2 mL), reflux, 40 h.
Scheme 2. The synthesis of substituted indolylquinazolinones 3. a All reactions were performed at 1.3 mmol scale of aldehyde 1 and 1.3 mmol of amide 2. Isolated yields. b The reaction was performed at 3 mmol of aldehyde 1 and 3 mmol of amide 2. Isolated yield. c The 2-(1H-indol-3-yl)quinazolin-4(3H)-one (3a) was isolated in 80–96% yield (n.d.—not detected). d The formation of indolylquinazolinones 3m, n were detected using thin-layer chromatography (TLC) and gas chromatography–mass spectrometry (GC/MS). The quinazolinone 5a was isolated in 37% and 59% yields. e The formation of indole (4a) was determined using TLC and GC/MS. f The corresponding N,N-dimethyl-1H-indole-3-carbothioamide (7a) was also isolated in a 21–50% yield. g The reaction conditions: aldehyde 1 (0.76 mmol), amide 2 (0.69 mmol), DMSO (1 mL), 100 °C, 3.5 h. Isolated yield. h The reaction conditions: aldehyde 1 (0.25 mmol), amide 2 (0.25 mmol), TFA (0.25 mmol), toluene (2 mL), reflux, 40 h.
Molecules 28 05348 sch002
Molecules 28 05348 sch003 550
Scheme 3. The functionalization of the N-atom of model indolylquinazolinone 3a.
Scheme 3. The functionalization of the N-atom of model indolylquinazolinone 3a.
Molecules 28 05348 sch003
Molecules 28 05348 sch004 550
Scheme 4. The functionalization of the amide moiety of model indolylquinazolinone 3a.
Scheme 4. The functionalization of the amide moiety of model indolylquinazolinone 3a.
Molecules 28 05348 sch004
Molecules 28 05348 g002 550
Figure 2. Influence of sublethal concentrations of amikacin (a) and 2-(5-iodo-1H-indol-3-yl)quinazolin-4(3H)-one (3k) (b) on the S. aureus ATCC 25923 biofilm biomass (OD570) and the number of colony-forming units (CFU) in plankton. Diagrams show the mean values of three (a) and four (b) experiments.
Figure 2. Influence of sublethal concentrations of amikacin (a) and 2-(5-iodo-1H-indol-3-yl)quinazolin-4(3H)-one (3k) (b) on the S. aureus ATCC 25923 biofilm biomass (OD570) and the number of colony-forming units (CFU) in plankton. Diagrams show the mean values of three (a) and four (b) experiments.
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Molecules 28 05348 g003 550
Figure 3. In vitro cytotoxicity of the compounds 3b, 3d–3g, k, and 3r to cells MCF7′, A549, HEK293T, and VA13 via MTT assay.
Figure 3. In vitro cytotoxicity of the compounds 3b, 3d–3g, k, and 3r to cells MCF7′, A549, HEK293T, and VA13 via MTT assay.
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Molecules 28 05348 g004 550
Figure 4. Best energy interactions between ligands and RelSeq protein synthetic domain active site. (a) Reference ligand indole-5-carboxylic acid; (b) 3o compound; and (c) 3ab compound. Protein chains are shown in grey thin sticks, ligands are shown in blue thick sticks, interactions are shown in dashed lines: yellow hydrogen bonds, blue pi–pi stacking and green pi-cation interactions, purple salt bridges.
Figure 4. Best energy interactions between ligands and RelSeq protein synthetic domain active site. (a) Reference ligand indole-5-carboxylic acid; (b) 3o compound; and (c) 3ab compound. Protein chains are shown in grey thin sticks, ligands are shown in blue thick sticks, interactions are shown in dashed lines: yellow hydrogen bonds, blue pi–pi stacking and green pi-cation interactions, purple salt bridges.
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Molecules 28 05348 g005 550
Figure 5. Best energy interactions between ligands and RelMtb protein synthetic domain active site. (a) 3b compound, (b) 3ab compound, and (c) 3p compound. Color coding is identical to Figure 4.
Figure 5. Best energy interactions between ligands and RelMtb protein synthetic domain active site. (a) 3b compound, (b) 3ab compound, and (c) 3p compound. Color coding is identical to Figure 4.
Molecules 28 05348 g005
Table
Table 1. Selected antimicrobial data (MIC and MBC/MFC, µg/mL) for the quinazolinone derivatives 3, 5a, and 6u a.
Table 1. Selected antimicrobial data (MIC and MBC/MFC, µg/mL) for the quinazolinone derivatives 3, 5a, and 6u a.
CompoundsC. a. 10231 bM. s. 70084 cE. c. 25922 dE. c. 8739 eS. a. 25923 fMRSA g
MICMFCMICMBCMICMBCMICMBCMICMBCMICMBC
3d125125- h---------
3g------------
3k7.807.80------3.907.800.983.90
3p250>500>500>500>500>500>500>500>500>500>500>500
3t125125----------
3u125500----------
3z>250>250>250>250>250>250>250>250>250>250>250>250
3ab500-----------
3ai62.50-----------
5a250500----------
6u125-----------
Cefotaximen.d. in.d.n.d.n.d.0.0380.0380.0380.0380.310.6119.5339.06
Cefazolinn.d.n.d.n.d.n.d.2.442.442.442.440.150.619.7739.06
Amikacinn.d.n.d.n.d.n.d.19.5319.5319.5319.534.889.779.779.77
Fluconazole1.947.8n.d.n.d.n.d.n.d.n.d.n.d.n.d.n.d.n.d.n.d.
Isoniazidn.d.n.d.4.589.16n.d.n.d.n.d.n.d.n.d.n.d.n.d.n.d.
Rifampicinn.d.n.d.1.2219.53n.d.n.d.n.d.n.d.n.d.n.d.n.d.n.d.
a In the table, the Mode values from 3–5 independent experiments are presented; b Candida albicans ATCC 10231; c Mycobacterium smegmatis ATCC 70084; d Escherichia coli ATCC 25922; e Escherichia coli ATCC 8739; f Staphylococcus aureus ATCC 25923; g Staphylococcus aureus ATCC 43300 (MRSA); h (-) >1000 µg/mL; i n.d.—not determined.
Table
Table 2. Activity of indolylquinazolinone 3k against Mycobacterium tuberculosis H37Rv.
Table 2. Activity of indolylquinazolinone 3k against Mycobacterium tuberculosis H37Rv.
Conc.,
µg/mL		Fluorescence Change (BACTEC MGIT 960 Growth System), Days
45678910111213141825303541
Control++++++++++++++++
100.0----------------
50.0----------------
25.0----------------
10.0----------------
5.0------------++++
2.5--++++++++++++++
1.0--++++++++++++++
Control—M. tuberculosis H37Rv inoculum without chemical compounds; - lack of growth; + growth of mycobacteria.
Table
Table 3. The IC50 or IC50abs (µM) for the cytotoxicity of the compounds 3b–3g, 3i, 3k, 3o, 3q–3ab, and 6u to cells MCF7′, A549, HEK293T, and VA13 via MTT assay a.
Table 3. The IC50 or IC50abs (µM) for the cytotoxicity of the compounds 3b–3g, 3i, 3k, 3o, 3q–3ab, and 6u to cells MCF7′, A549, HEK293T, and VA13 via MTT assay a.
CompoundsMCF7′VA13A549HEK293T
IC50IC50absIC50IC50absIC50IC50absIC50IC50abs
3b2.1 ± 0.427.1 ± 4.711.4 ± 1.229.4 ± 4.32.1 ± 0.33.6 ± 0.46.2 ± 0.78.3 ± 0.7
3c4.1 ± 0.610.9 ± 1.13.7 ± 0.44.0 ± 0.23.7 ± 0.41.7 ± 0.21.8 ± 0.73.4 ± 0.6
3dle bx > 10047.0 ± 11.869.3 ± 3.143.0 ± 5.140.5 ± 2.75.5 ± 0.956.8 ± 7.9
3ex > 100x > 100x > 100x > 100~18.0 ± 2.3~1x > 100
3f4.9 ± 0.77.1 ± 0.83.4 ± 0.43.8 ± 0.30,96 ± 0,181.3 ± 0.090.95 ± 0.281.9 ± 0.16
3g0.57 ± 0.0527.6 ± 7.40.84 ± 0.3813.5 ± 3.60.75 ± 0.073.3 ± 0,60.73 ± 0.159.8 ± 2.5
3i11.8 ± 0.710.9 ± 0.66.6 ± 0.57.1 ± 0.411.7± 1.29.0 ± 0.73.4 ± 0.24.1 ± 0.3
3k5.9 ± 0.36.1 ± 0.37.9 ± 0.48.5 ± 0.36.5 ± 0.26.5± 0.23.4 ± 0.13.5 ± 0.1
3on.a. c57.1 ± 6.2~2067.8 ± 21.19.1 ± 1.238.1 ± 6.4n.a.19.8 ± 3.4
3q~2019.7 ± 2.516.5 ± 2.523.2 ± 2.96.1 ± 1.16.9 ± 0.68.2 ± 1.19.0 ± 0.7
3r5.4 ± 2.2x > 1002.5 ± 0.760.1 ± 23.03.5 ± 0327.6 ± 4.91.8 ± 0.448.9 ± 13.6
3s13.6 ± 1.210.1 ± 1.013.5 ± 1.812.9 ± 0.89.4 ± 039.3 ± 0.312.4 ± 0.512,7 ± 0.4
3tx > 33.3x > 33.3x > 33.3x > 33.3x > 333x > 33.3x > 33.3x > 33.3
3u0.30 ± 0.14 (le)x > 100x > 100x > 100n.a.107.4 ± 27.7n.a.66.8 ± 3.6
3vn.a.93.0 ± 3.1n.a.80.6 ± 3.9n.a.57.0 ± 3.9n.a.62.9 ± 2.8
3w0.53 ± 0.44 (le)x > 100x > 100115.8 ± 0.2n.a.105.1 ± 15.8n.a.104.8 ± 2.1
3xlex > 100lex > 100lex > 10057.3 ± 34.5152.3 ± 30.3
3yx > 100x > 100x > 100x > 10061.4 ± 58.1x > 100x > 100x > 100
3z8.4 ± 0.7016.1 ± 0.93.0 ± 0.44.2 ± 0.42.6 ± 0.33.2 ± 0.33.9 ± 0.14.0 ± 0.1
3aax > 100x > 100x > 100163.4 ± 19.7x > 10065.5 ± 5.8x > 100195.8 ± 51.0
3abx > 100x > 10044.9 ± 11.790.8 ± 8.7n.a.169.1 ± 47.230.5 ± 1.132.2 ± 1.6
6ux > 100x > 100x > 100129.3 ± 23.4x > 100215.7 ± 35.8x > 100x > 100
doxorubicin0.195 ± 0.010.195 ± 0.010.087 ± 0.040.087 ± 0.040.038 ± 0.010.038 ± 0.010.018 ± 0.0010.018 ± 0.001
a Data over 100 µM—extrapolation result; b le—the effect does not reach 50%, but there is some cytotoxicity; c n.a.—not active.
Table
Table 4. Docking results for the compounds 3a–3g, 3k, 3l, 3o–3r, 3ab, 3ah, 3am, 3ak, and 7a, 7b into RelSeq and RelMtb models reported as docking scores.
Table 4. Docking results for the compounds 3a–3g, 3k, 3l, 3o–3r, 3ab, 3ah, 3am, 3ak, and 7a, 7b into RelSeq and RelMtb models reported as docking scores.
RelSeq SYN Active SiteRelMtb SYN Active Site
LigandDocking Score (kcal mol−1)LigandDocking Score (kcal mol−1)
Indole-5-carboxylic acid (COO−)−6.423b−7.61
3o−4.763ab−7.43
3ab−4.723p−6.71
3a−4.643a−6.70
3b−4.533ah−6.67
3d−4.483r−6.66
3e−4.483am−6.62
3ak−4.453o−6.56
7a−4.39Indole-5-carboxylicacid (COO−)−6.55
3ah−4.347a−6.52
3r−4.333l−6.51
3p−4.32DMNP a (COO−)−6.41
DMNP a (COO−)−4.283k−6.17
3q−4.283c−6.10
3g−4.153d−6.07
3c−4.113q−6.05
3l−4.053ai−5.82
3i−4.013f−5.77
3ai−3.973i−5.57
3f−3.843ak−5.50
3am−3.783g−5.33
3k−3.777b−4.96
7b−3.733e−4.25
a DMNP—4-(4,7-dimethyl-1,2,3,4-tetrahydronaphthalen-1-yl)pentanoic acid.
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Mendogralo, E.Y.; Nesterova, L.Y.; Nasibullina, E.R.; Shcherbakov, R.O.; Tkachenko, A.G.; Sidorov, R.Y.; Sukonnikov, M.A.; Skvortsov, D.A.; Uchuskin, M.G. The Synthesis and Biological Evaluation of 2-(1H-Indol-3-yl)quinazolin-4(3H)-One Derivatives. Molecules 2023, 28, 5348. https://doi.org/10.3390/molecules28145348

AMA Style

Mendogralo EY, Nesterova LY, Nasibullina ER, Shcherbakov RO, Tkachenko AG, Sidorov RY, Sukonnikov MA, Skvortsov DA, Uchuskin MG. The Synthesis and Biological Evaluation of 2-(1H-Indol-3-yl)quinazolin-4(3H)-One Derivatives. Molecules. 2023; 28(14):5348. https://doi.org/10.3390/molecules28145348

Chicago/Turabian Style

Mendogralo, Elena Y., Larisa Y. Nesterova, Ekaterina R. Nasibullina, Roman O. Shcherbakov, Alexander G. Tkachenko, Roman Y. Sidorov, Maxim A. Sukonnikov, Dmitry A. Skvortsov, and Maxim G. Uchuskin. 2023. "The Synthesis and Biological Evaluation of 2-(1H-Indol-3-yl)quinazolin-4(3H)-One Derivatives" Molecules 28, no. 14: 5348. https://doi.org/10.3390/molecules28145348

APA Style

Mendogralo, E. Y., Nesterova, L. Y., Nasibullina, E. R., Shcherbakov, R. O., Tkachenko, A. G., Sidorov, R. Y., Sukonnikov, M. A., Skvortsov, D. A., & Uchuskin, M. G. (2023). The Synthesis and Biological Evaluation of 2-(1H-Indol-3-yl)quinazolin-4(3H)-One Derivatives. Molecules, 28(14), 5348. https://doi.org/10.3390/molecules28145348

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