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       The synthon 3-(anthracen-9-yl)-2-cyanoacryloyl chloride 4 was synthesized and used to synthesize a variety of highly active heterocyclic compounds through its reaction with various nitrogen nucleophiles. The structure of each synthesized heterocyclic compound was thoroughly characterized using spectroscopic and elemental analysis. Ten of the thirteen novel heterocyclic compounds showed encouraging efficacy against multidrug-resistant bacteria (MRSA). Among them, compounds 6, 7, 10, 13b, and 14 showed the highest antibacterial activity with inhibition zones close to 4 cm. However, molecular docking studies revealed that the compounds had different binding affinities to penicillin-binding protein 2a (PBP2a), a key target for MRSA resistance. Some compounds such as 7, 10 and 14 showed higher binding affinity and interaction stability at the active site of PBP2a compared to the co-crystallized quinazolinone ligand. In contrast, compounds 6 and 13b had lower docking scores but still exhibited significant antibacterial activity, with compound 6 having the lowest MIC (9.7 μg/100 μL) and MBC (78.125 μg/100 μL) values. Docking analysis revealed key interactions including hydrogen bonding and π-stacking, particularly with residues such as Lys 273, Lys 316 and Arg 298, which were identified as interacting with the co-crystallized ligand in the crystal structure of PBP2a. These residues are essential for the enzymatic activity of PBP2a. These results suggest that the synthesized compounds may serve as promising anti-MRSA drugs, highlighting the importance of combining molecular docking with bioassays to identify effective therapeutic candidates.
       In the first few years of this century, research efforts were mainly focused on developing new, simple procedures and methods for the synthesis of several innovative heterocyclic systems with antimicrobial activity using readily available starting materials.
       Acrylonitrile moieties are considered as important starting materials for the synthesis of many remarkable heterocyclic systems because they are highly reactive compounds. Moreover, 2-cyanoacryloyl chloride derivatives have been widely used in recent years for the development and synthesis of products of vital importance in the field of pharmacological applications, such as drug intermediates1,2,3, precursors of anti-HIV, antiviral, anticancer, antibacterial, antidepressant and antioxidant agents4,5,6,7,8,9,10. Recently, the biological efficacy of anthracene and its derivatives, including their antibiotic, anticancer11,12, antibacterial13,14,15 and insecticidal properties16,17, have attracted much attention18,19,20,21. The antimicrobial compounds containing acrylonitrile and anthracene moieties are shown in Figures 1 and 2.
       According to the World Health Organization (WHO) (2021), antimicrobial resistance (AMR) is a global threat to health and development22,23,24,25. Patients cannot be cured, resulting in longer hospital stays and the need for more expensive drugs, as well as increased mortality and disability. The lack of effective antimicrobials often leads to treatment failure for various infections, especially during chemotherapy and major surgeries.
       According to the World Health Organization 2024 report, methicillin-resistant Staphylococcus aureus (MRSA) and E. coli are included in the list of priority pathogens. Both bacteria are resistant to many antibiotics, so they represent infections that are difficult to treat and control, and there is an urgent need to develop new and effective antimicrobial compounds to address this problem. Anthracene and its derivatives are well-known antimicrobials that can act on both Gram-positive and Gram-negative bacteria. The aim of this study is to synthesize a new derivative that can combat these pathogens that are dangerous to health.
       The World Health Organization (WHO) reports that many bacterial pathogens are resistant to multiple antibiotics, including methicillin-resistant Staphylococcus aureus (MRSA), a common cause of infection in the community and health care settings. Patients with MRSA infections are reported to have a 64% higher mortality rate than those with drug-susceptible infections. In addition, E. coli poses a global risk because the last line of defense against carbapenem-resistant Enterobacteriaceae (i.e., E. coli) is colistin, but colistin-resistant bacteria have recently been reported in several countries. 22,23,24,25
       Therefore, according to the World Health Organization Global Action Plan on Antimicrobial Resistance26, there is an urgent need for the discovery and synthesis of new antimicrobials. The great potential of anthracene and acrylonitrile as antibacterial27, antifungal28, anticancer29 and antioxidant30 agents has been highlighted in numerous published papers. In this regard, it can be said that these derivatives are good candidates for use against methicillin-resistant Staphylococcus aureus (MRSA).
       Previous literature reviews motivated us to synthesize new derivatives in these classes. Therefore, the present study aimed to develop novel heterocyclic systems containing anthracene and acrylonitrile moieties, evaluate their antimicrobial and antibacterial efficacy, and investigate their potential binding interactions with penicillin-binding protein 2a (PBP2a) by molecular docking. Building on the previous studies, the present study continued the synthesis, biological evaluation, and computational analysis of heterocyclic systems to identify promising antimethicillin-resistant Staphylococcus aureus (MRSA) agents with potent PBP2a inhibitory activity31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49.
       Our current research focuses on the synthesis and antimicrobial evaluation of novel heterocyclic compounds containing anthracene and acrylonitrile moieties. 3-(anthracen-9-yl)-2-cyanoacryloyl chloride 4 was prepared and used as a building block for the construction of novel heterocyclic systems.
       The structure of compound 4 was determined using spectral data. The 1H-NMR spectrum showed the presence of CH= at 9.26 ppm, the IR spectrum showed the presence of a carbonyl group at 1737 cm−1 and a cyano group at 2224 cm−1, and the 13CNMR spectrum also confirmed the proposed structure (see Experimental section).
       The synthesis of 3-(anthracen-9-yl)-2-cyanoacryloyl chloride 4 was accomplished by hydrolysis of aromatic groups 250, 41, 42, 53 with ethanolic sodium hydroxide solution (10%) to give acids 354, 45, 56, which were then treated with thionyl chloride on a water bath to give acryloyl chloride derivative 4 in high yield (88.5%), as shown in Figure 3.
       To create new heterocyclic compounds with the expected antibacterial efficacy, the reaction of acyl chloride 4 with various dinucleophiles was carried out.
       The acid chloride 4 was treated with hydrazine hydrate at 0° for one hour. Unfortunately, pyrazolone 5 was not obtained. The product was an acrylamide derivative whose structure was confirmed by spectral data. Its IR spectrum showed absorption bands of C=O at 1720 cm−1, C≡N at 2228 cm−1 and NH at 3424 cm−1. The 1H-NMR spectrum showed an exchange singlet signal of the olefin protons and NH protons at 9.3 ppm (see Experimental Section).
       Two moles of acid chloride 4 were reacted with one mole of phenylhydrazine to afford N-phenylacryloylhydrazine derivative 7 in good yield (77%) (Figure 5). The structure of 7 was confirmed by infrared spectroscopy data, which showed absorption of two C=O groups at 1691 and 1671 cm−1, absorption of CN group at 2222 cm−1 and absorption of NH group at 3245 cm−1, and its 1H-NMR spectrum showed CH group at 9.15 and 8.81 ppm and NH proton at 10.88 ppm (see Experimental section).
       In this study, the reaction of acyl chloride 4 with 1,3-dinucleophiles was investigated. Treatment of acyl chloride 4 with 2-aminopyridine in 1,4-dioxane with TEA as base at room temperature afforded acrylamide derivative 8 (Figure 5), the structure of which was identified using spectral data. IR spectra showed absorption bands of cyano stretching at 2222 cm−1, NH at 3148 cm−1, and carbonyl at 1665 cm−1; 1H NMR spectra confirmed the presence of olefin protons at 9.14 ppm (see Experimental Section).
       Compound 4 reacts with thiourea to give pyrimidinethione 9; compound 4 reacts with thiosemicarbazide to give thiopyrazole derivative 10 (Figure 5). The structures of compounds 9 and 10 were confirmed by spectral and elemental analysis (see Experimental section).
       Tetrazine-3-thiol 11 was prepared by the reaction of compound 4 with thiocarbazide as a 1,4-dinucleophile (Figure 5), and its structure was confirmed by spectroscopy and elemental analysis. In the infrared spectrum, the C=N bond appeared at 1619 cm−1. At the same time, its 1H-NMR spectrum retained multiplate signals of aromatic protons at 7.78–8.66 ppm and SH protons at 3.31 ppm (see Experimental Section).
       Acryloyl chloride 4 reacts with 1,2-diaminobenzene, 2-aminothiophenol, anthranilic acid, 1,2-diaminoethane, and ethanolamine as 1,4-dinucleophiles to form new heterocyclic systems (13–16).
       The structures of these newly synthesized compounds were confirmed by spectral and elemental analysis (see Experimental section). 2-Hydroxyphenylacrylamide derivative 17 was obtained by reaction with 2-aminophenol as a dinucleophile (Figure 6), and its structure was confirmed by spectral and elemental analysis. The infrared spectrum of compound 17 showed that the C=O and C≡N signals appeared at 1681 and 2226 cm−1, respectively. Meanwhile, its 1H-NMR spectrum retained the singlet signal of the olefin proton at 9.19 ppm, and the OH proton appeared at 9.82 ppm (see Experimental section).
       The reaction of acid chloride 4 with one nucleophile (e.g., ethylamine, 4-toluidine, and 4-methoxyaniline) in dioxane as a solvent and TEA as a catalyst at room temperature afforded green crystalline acrylamide derivatives 18, 19a, and 19b. Elemental and spectral data of compounds 18, 19a, and 19b confirmed the structures of these derivatives (see Experimental Section) (Figure 7).
       After screening the antimicrobial activity of various synthetic compounds, different results were obtained as shown in Table 1 and Figure 8 (see figure file). All the tested compounds showed different degrees of inhibition against the Gram-positive bacterium MRSA, while the Gram-negative bacterium Escherichia coli showed complete resistance to all the compounds. The tested compounds can be divided into three categories based on the diameter of the inhibition zone against MRSA. The first category was the most active and consisted of five compounds (6, 7, 10, 13b and 14). The diameter of the inhibition zone of these compounds was close to 4 cm; the most active compounds in this category were compounds 6 and 13b. The second category was moderately active and consisted of another five compounds (11, 13a, 15, 18 and 19a). The inhibition zone of these compounds ranged from 3.3 to 3.65 cm, with compound 11 showing the largest inhibition zone of 3.65 ± 0.1 cm. On the other hand, the last group contained three compounds (8, 17 and 19b) with the lowest antimicrobial activity (less than 3 cm). Figure 9 shows the distribution of the different inhibition zones.
       Further investigation of the antimicrobial activity of the tested compounds involved determination of the MIC and MBC for each compound. The results varied slightly (as shown in Tables 2, 3 and Figure 10 (see figure file)), with compounds 7, 11, 13a and 15 apparently being reclassified as the best compounds. They had the same lowest MIC and MBC values ​​(39.06 μg/100 μL). Although compounds 7 and 8 had lower MIC values ​​(9.7 μg/100 μL), their MBC values ​​were higher (78.125 μg/100 μL). Therefore, they were considered weaker than the previously mentioned compounds. However, these six compounds were the most effective of those tested, as their MBC values ​​were below 100 μg/100 μL.
       Compounds (10, 14, 18 and 19b) were less active compared to other tested compounds as their MBC values ​​ranged from 156 to 312 μg/100 μL. On the other hand, compounds (8, 17 and 19a) were the least promising as they had the highest MBC values ​​(625, 625 and 1250 μg/100 μL, respectively).
       Finally, according to the tolerance levels shown in Table 3, the tested compounds can be divided into two categories based on their mode of action: compounds with bactericidal effect (7, 8, 10, 11, 13a, 15, 18, 19b) and compounds with antibacterial effect (6, 13b, 14, 17, 19a). Among them, compounds 7, 11, 13a and 15 are preferred, which exhibit killing activity at a very low concentration (39.06 μg/100 μL).
       Ten of the thirteen compounds tested showed potential against antibiotic-resistant methicillin-resistant Staphylococcus aureus (MRSA). Therefore, further screening with more antibiotic-resistant pathogens (especially local isolates covering pathogenic Gram-positive and Gram-negative bacteria) and pathogenic yeasts is recommended, as well as cytotoxic testing of each compound to assess its safety.
       Molecular docking studies were conducted to evaluate the potential of the synthesized compounds as inhibitors of penicillin-binding protein 2a (PBP2a) in methicillin-resistant Staphylococcus aureus (MRSA). PBP2a is a key enzyme involved in bacterial cell wall biosynthesis, and inhibition of this enzyme interferes with cell wall formation, ultimately leading to bacterial lysis and cell death1. The docking results are listed in Table 4 and described in more detail in the supplementary data file, and the results show that several compounds exhibited strong binding affinity for PBP2a, particularly key active site residues such as Lys 273, Lys 316, and Arg 298. The interactions, including hydrogen bonding and π-stacking, were very similar to those of the co-crystallized quinazolinone ligand (CCL), indicating the potential of these compounds as potent inhibitors.
       The molecular docking data, along with other computational parameters, strongly suggested that PBP2a inhibition was the key mechanism responsible for the observed antibacterial activity of these compounds. The docking scores and root mean square deviation (RMSD) values ​​further revealed the binding affinity and stability, supporting this hypothesis. As shown in Table 4, while several compounds showed good binding affinity, some compounds (e.g., 7, 9, 10, and 14) had higher docking scores than the co-crystallized ligand, indicating that they may have stronger interactions with the active site residues of PBP2a. However, the most bioactive compounds 6 and 13b showed slightly lower docking scores (-5.98 and -5.63, respectively) compared to the other ligands. This suggests that although docking scores can be used to predict binding affinity, other factors (e.g., ligand stability and molecular interactions in the biological environment) also play a key role in determining antibacterial activity. Notably, the RMSD values ​​of all synthesized compounds were below 2 Å, confirming that their docking poses are structurally consistent with the binding conformation of the co-crystallized ligand, further supporting their potential as potent PBP2a inhibitors.
       Although docking scores and RMS values ​​provide valuable predictions, the correlation between these docking results and antimicrobial activity is not always clear at first glance. Although PBP2a inhibition is strongly supported as a key factor influencing antimicrobial activity, several differences suggest that other biological properties also play an important role. Compounds 6 and 13b showed the highest antimicrobial activity, with both an inhibition zone diameter of 4 cm and the lowest MIC (9.7 μg/100 μL) and MBC (78.125 μg/100 μL) values, despite their lower docking scores compared to compounds 7, 9, 10 and 14. This suggests that although PBP2a inhibition contributes to antimicrobial activity, factors such as solubility, bioavailability and interaction dynamics in the bacterial environment also influence overall activity. Figure 11 shows their docking poses, indicating that both compounds, even with relatively low binding scores, are still able to interact with key residues of PBP2a, potentially stabilizing the inhibition complex. This highlights that while molecular docking provides important insights into PBP2a inhibition, other biological factors must be considered to fully understand the real-world antimicrobial effects of these compounds.
       Using the crystal structure of PBP2a (PDB ID: 4CJN), 2D and 3D interaction maps of the most active compounds 6 and 13b docked with penicillin-binding protein 2a (PBP2a) of methicillin-resistant Staphylococcus aureus (MRSA) were constructed. These maps compare the interaction patterns of these compounds with the re-docked co-crystallized quinazolinone ligand (CCL), highlighting key interactions such as hydrogen bonding, π-stacking, and ionic interactions.
       A similar pattern was observed for compound 7, which showed a relatively high docking score (-6.32) and a similar inhibition zone diameter (3.9 cm) to compound 10. However, its MIC (39.08 μg/100 μL) and MBC (39.06 μg/100 μL) were significantly higher, indicating that it required higher concentrations to exhibit antibacterial effect. This suggests that although compound 7 showed strong binding affinity in docking studies, factors such as bioavailability, cellular uptake, or other physicochemical properties may limit its biological efficacy. Although compound 7 showed bactericidal properties, it was less effective in inhibiting bacterial growth compared to compounds 6 and 13b.
       Compound 10 showed a more dramatic difference with the highest docking score (-6.40), indicating strong binding affinity to PBP2a. However, its zone of inhibition diameter (3.9 cm) was comparable to compound 7, and its MBC (312 μg/100 μL) was significantly higher than compounds 6, 7, and 13b, indicating weaker bactericidal activity. This suggests that despite good docking predictions, compound 10 was less effective in killing MRSA due to other limiting factors such as solubility, stability, or poor permeability of the bacterial membrane. These results support the understanding that while PBP2a inhibition plays a key role in antibacterial activity, it does not fully explain the differences in biological activity observed among the tested compounds. These differences suggest that further experimental analyses and in-depth biological evaluations are needed to fully elucidate the antibacterial mechanisms involved.
       The molecular docking results in Table 4 and the Supplementary Data File highlight the complex relationship between docking scores and antimicrobial activity. Although compounds 6 and 13b have lower docking scores than compounds 7, 9, 10, and 14, they exhibit the highest antimicrobial activity. Their interaction maps (shown in Figure 11) indicate that despite their lower binding scores, they still form significant hydrogen bonds and π-stacking interactions with key residues of PBP2a that can stabilize the enzyme-inhibitor complex in a biologically beneficial manner. Despite the relatively low docking scores of 6 and 13b, their enhanced antimicrobial activity suggests that other properties such as solubility, stability, and cellular uptake should be considered in conjunction with the docking data when assessing inhibitor potential. This highlights the importance of combining docking studies with experimental antimicrobial analysis to accurately assess the therapeutic potential of new compounds.
       These results highlight that while molecular docking is a powerful tool for predicting binding affinity and identifying potential mechanisms of inhibition, it should not be relied upon alone to determine antimicrobial efficacy. The molecular data suggest that PBP2a inhibition is a key factor influencing antimicrobial activity, but changes in biological activity suggest that other physicochemical and pharmacokinetic properties must be optimized to enhance therapeutic efficacy. Future studies should focus on optimizing the chemical structure of compounds 7 and 10 to improve bioavailability and cellular uptake, ensuring that strong docking interactions are translated into actual antimicrobial activity. Further studies, including additional bioassays and structure-activity relationship (SAR) analysis, will be critical to further our understanding of how these compounds function as PBP2a inhibitors and to develop more effective antimicrobial agents.
       Compounds synthesized from 3-(anthracen-9-yl)-2-cyanoacryloyl chloride 4 exhibited varying degrees of antimicrobial activity, with several compounds demonstrating significant inhibition of methicillin-resistant Staphylococcus aureus (MRSA). Structure-activity relationship (SAR) analysis revealed key structural features underlying the antimicrobial efficacy of these compounds.
       The presence of both acrylonitrile and anthracene groups proved to be critical for enhancing antimicrobial activity. The highly reactive nitrile group in acrylonitrile is necessary to facilitate interactions with bacterial proteins, thereby contributing to the antimicrobial properties of the compound. Compounds containing both acrylonitrile and anthracene consistently demonstrated stronger antimicrobial effects. The aromaticity of the anthracene group further stabilized these compounds, potentially enhancing their biological activity.
       The introduction of heterocyclic rings significantly improved the antibacterial efficacy of several derivatives. In particular, benzothiazole derivative 13b and acrylhydrazide derivative 6 showed the highest antibacterial activity with an inhibition zone of approximately 4 cm. These heterocyclic derivatives showed more significant biological effects, indicating that the heterocyclic structure plays a key role in the antibacterial effects. Likewise, pyrimidinethione in compound 9, thiopyrazole in compound 10, and tetrazine ring in compound 11 contributed to the antibacterial properties of the compounds, further highlighting the importance of heterocyclic modification.
       Among the synthesized compounds, 6 and 13b stood out for their excellent antibacterial activities. The minimum inhibitory concentration (MIC) of compound 6 was 9.7 μg/100 μL, and the minimum bactericidal concentration (MBC) was 78.125 μg/100 μL, highlighting its excellent ability to clear methicillin-resistant Staphylococcus aureus (MRSA). Similarly, compound 13b had an inhibition zone of 4 cm and low MIC and MBC values, confirming its potent antibacterial activity. These results highlight the key roles of acrylohydrazide and benzothiazole functional groups in determining the bioefficacy of these compounds.
       In contrast, compounds 7, 10, and 14 exhibited moderate antibacterial activity with inhibition zones ranging from 3.65 to 3.9 cm. These compounds required higher concentrations to completely kill the bacteria, as reflected by their relatively high MIC and MBC values. Although these compounds were less active than compounds 6 and 13b, they still showed significant antibacterial potential, suggesting that the incorporation of acrylonitrile and anthracene moieties into the heterocyclic ring contributes to their antibacterial effect.
       The compounds have different modes of action, some exhibiting bactericidal properties and others exhibiting bacteriostatic effects. Compounds 7, 11, 13a, and 15 are bactericidal and require lower concentrations to completely kill bacteria. In contrast, compounds 6, 13b, and 14 are bacteriostatic and can inhibit bacterial growth at lower concentrations, but require higher concentrations to completely kill bacteria.
       Overall, the structure-activity relationship analysis highlights the importance of introducing acrylonitrile and anthracene moieties and heterocyclic structures to achieve significant antibacterial activity. These results suggest that optimization of these structural components and exploration of further modifications to improve solubility and membrane permeability may lead to the development of more effective anti-MRSA drugs.
       All reagents and solvents were purified and dried using standard procedures (El Gomhouria, Egypt). Melting points were determined using a GallenKamp electronic melting point apparatus and are reported without correction. Infrared (IR) spectra (cm⁻1) were recorded at the Department of Chemistry, Faculty of Science, Ain Shams University using potassium bromide (KBr) pellets on a Thermo Electron Nicolet iS10 FTIR spectrometer (Thermo Fisher Scientific, Waltham, MA, USA).
       1H NMR spectra were obtained at 300 MHz using a GEMINI NMR spectrometer (GEMINI Manufacturing & Engineering, Anaheim, CA, USA) and a BRUKER 300 MHz NMR spectrometer (BRUKER Manufacturing & Engineering, Inc.). Tetramethylsilane (TMS) was used as an internal standard with deuterated dimethyl sulfoxide (DMSO-d₆). NMR measurements were performed at the Faculty of Science, Cairo University, Giza, Egypt. Elemental analysis (CHN) was performed using a Perkin-Elmer 2400 Elemental Analyzer and the results obtained are in good agreement with the calculated values.
       A mixture of acid 3 (5 mmol) and thionyl chloride (5 ml) was heated in a water bath at 65 °C for 4 h. Excess thionyl chloride was removed by distillation under reduced pressure. The resulting red solid was collected and used without further purification. Melting point: 200-202 °C, yield: 88.5%. IR (KBr, ν, cm−1): 2224 (C≡N), 1737 (C=O). 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 9.26 (s, 1H, CH=), 7.27-8.57 (m, 9H, heteroaromatization). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 115.11 (C≡N), 124.82–130.53 (CH anthracene), 155.34, 114.93 (CH=C–C=O), 162.22 (C=O); HRMS (ESI) m/z [M + H]+: 291.73111. Analyst. Calculated for C18H10ClNO (291.73): C, 74.11; H, 3.46; N, 4.80. Found: C, 74.41; H, 3.34; N, 4.66%.
       At 0°C, 4 (2 mmol, 0.7 g) was dissolved in anhydrous dioxane (20 ml) and hydrazine hydrate (2 mmol, 0.16 ml, 80%) was added dropwise and stirred for 1 h. The precipitated solid was collected by filtration and recrystallized from ethanol to give compound 6.
       Green crystals, melting point 190-192℃, yield 69.36%; IR (KBr) ν=3424 (NH), 2228 (C≡N), 1720 (C=O), 1621 (C=N) cm−1. 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 9.3 (br s, H, NH, exchangeable), 7.69-8.51 (m, 18H, heteroaromatic), 9.16 (s, 1H, CH=), 8.54 (s, 1H, CH=); Calculated value for C33H21N3O (475.53): C, 83.35; H, 4.45; N, 8.84. Found: C, 84.01; H, 4.38; N, 8.05%.
       Dissolve 4 (2 mmol, 0.7 g) in 20 ml of anhydrous dioxane solution (containing a few drops of triethylamine), add phenylhydrazine/2-aminopyridine (2 mmol) and stir at room temperature for 1 and 2 h, respectively. Pour the reaction mixture into ice or water and acidify with dilute hydrochloric acid. Filter off the separated solid and recrystallize from ethanol to obtain 7 and recrystallize from benzene to obtain 8.
       Green crystals, melting point 160-162℃, yield 77%; IR (KBr, ν, cm−1): 3245 (NH), 2222 (C≡N), 1691 (C=O), 1671 (C=O) cm−1. 1H-NMR (400 MHz, DMSO-d6): δ (ppm): 10.88 (s, 1H, NH, exchangeable), 9.15 (s, 1H, CH=), 8.81 (s, 1H, CH=), 6.78-8.58 (m, 23H, heteroaromatic); Calculated value for C42H26N4O2 (618.68): C, 81.54; H, 4.24; N, 9.06. Found: C, 81.96; H, 3.91; N, 8.91%.
       4 (2 mmol, 0.7 g) was dissolved in 20 ml of anhydrous dioxane solution (containing a few drops of triethylamine), 2-aminopyridine (2 mmol, 0.25 g) was added and the mixture was stirred at room temperature for 2 h. The reaction mixture was poured into ice water and acidified with dilute hydrochloric acid. The formed precipitate was filtered off and recrystallized from benzene, giving green crystals of 8 with a melting point of 146-148 °C and a yield of 82.5%; infrared spectrum (KBr) ν: 3148 (NH), 2222 (C≡N), 1665 (C=O) cm−1. 1H NMR (400 MHz, DMSO-d6): δ (ppm): 8.78 (s, H, NH, exchangeable), 9.14 (s, 1H, CH=), 7.36-8.55 (m, 13H, heteroaromatization); Calculated for C23H15N3O (348.38): C, 79.07; H, 4.33; N, 12.03. Found: C, 78.93; H, 3.97; N, 12.36%.
       Compound 4 (2 mmol, 0.7 g) was dissolved in 20 ml of dry dioxane (containing a few drops of triethylamine and 2 mmol of thiourea/semicarbazide) and heated under reflux for 2 h. The solvent was evaporated in vacuo. The residue was recrystallized from dioxane to give a mixture.