231-237In the present paper, the synthesis, characterization, antimicrobial activity and in silico molecular docking study of 6-((arylidene)amino)-4-(4-chlorophenyl)-2-oxo-1-((1-(2-oxo-2H-chromen-3-yl)ethylidene)amino)-1,2-dihydropyridine-3,5-dicarbonitriles 4a-o have been reported. Compounds 4d, 4g, 4j, 4k, 4m and 4o show significant activity. Structure determination of the synthesized compounds has been done by the standard spectroscopic techniques. It is observed that biological activity is influenced by electronic environment of the molecules. Electron withdrawing group at para position plays a major role for enhancing the biological activity for antibacterial activity and the electron donating group at para position for antifungal activity. Compounds 4a-o have been further evaluated for cytotoxicity on HeLa cells. From the cytotoxicity results, compounds have been found to possess low cytotoxicity with potent antimicrobial activity

Desai, N C

Jadeja, Krunalsinh A

Joshi, Surbhi B

Karkar, Tushar J

Vaja, Darshita V

Vekariya, Rajesh H

English

NOPR

Indian Journal of Chemistry Vol. 59B, February 2020, pp.  231-237       Synthesis, characterization, molecular docking studies and biological activity of coumarin linked 2-pyridone heterocycles N C Desai*a, Tushar J Karkarb, Rajesh H Vekariyac, Surbhi B Joshia, Krunalsinh A Jadejaa & Darshita V Vajaa a Division of Medicinal Chemistry, Department of Chemistry, Mahatma Gandhi Campus Maharaja Krishnakumarsinhji Bhavnagar University, Bhavnagar 364 002, India (DST-FIST Sponsored & UGC NON-SAP) b C B Patel Computer College and JNM Patel Science College (DRB), New City light Road, Bharthana (Vesu), Surat 395 007, India c Research Scientist, Piramal Pharma Solutions, Plot No. 18, Pharmez, Matoda Village, Ahmedabad 382 220, India E-mail: dnisheeth@rediffmail.com Received 30 August 2019; accepted (revised) 16 December 2019 In the present paper, the synthesis, characterization, antimicrobial activity and in silico molecular docking study of  6-((arylidene)amino)-4-(4-chlorophenyl)-2-oxo-1-((1-(2-oxo-2H-chromen-3-yl)ethylidene)amino)-1,2-dihydropyridine- 3,5-dicarbonitriles 4a-o have been reported. Compounds 4d, 4g, 4j, 4k, 4m and 4o show significant activity. Structure determination of the synthesized compounds has been done by the standard spectroscopic techniques. It is observed that biological activity is influenced by electronic environment of the molecules. Electron withdrawing group at para position plays a major role for enhancing the biological activity for antibacterial activity and the electron donating group at para position for antifungal activity. Compounds 4a-o have been further evaluated for cytotoxicity on HeLa cells. From the cytotoxicity results, compounds have been found to possess low cytotoxicity with potent antimicrobial activity. Keywords: Medicinal chemistry, antibacterial, antifungal, cytotoxicity, molecular docking The synthesis and biological activities of coumarin based 2-pyridone derivatives 4a-o occupied an important position in heterocyclic chemistry as well as in medicinal chemistry. Coumarin derivatives displayed a wide range of medicinal activity1-6. Moreover, these derivatives were also found to possess very good antimicrobial activity as shown in the literature7-11. They are responsible for positive activity of several types of cancer including malignant melanoma, renal cell carcinoma, leukemia, prostate and breast cancer cells progression with less cytotoxicity12-15. Many currently available antimicrobial agents possessed coumarin skeleton such as Chlorobiocin and Novobiocin16 in their molecular frame work. 2-Pyridones denote versatile class of pharmacophores having very good therapeutic effect17 on several diseases. Heterocyclic compounds having 2-pyridone moiety showed various biological activities, such as analgesic18, anti-HIV19 and antitumoral20. The most prominent examples of prescribed agents featuring the 2-pyridone  nucleus include the Phosphodiester (PDE) III inhibitor Amrinone, Antifungal Ciclopirox, Cardiotonic agent Olprinone, Antitumor antibiotic Diazaquinomycin A, HIV-1 Inhibitors Pyridinone L as in shown Figure 1. In this paper, we have adopted the concept of Michael addition having α-β unsaturated carbonyl attack followed by hydrolytic cyclization with subsequent oxidative aromatization is the most common method used to synthesize 2-pyridone heterocycles21-23. In continuation of our previous work for searching novel pharmacophores with potent antimicrobial activity24-30, we have designed novel compounds having coumarin and 2-pyridone in a single molecular framework. Newly synthesized compounds were tested for antimicrobial potency and also evaluated cytotoxicity on HeLa cell line. Structural elucidation of newly synthesized compounds 4a-o was carried out by different analytical techniques like IR, NMR and mass spectrometry.  Results and Discussion  Chemistry The synthetic pathway of the titled compounds is given in Scheme I. 3-acetyl-2H-chromen-2-one (1) was synthesized with salicylaldehyde (A) and ethyl acetoacetate (B). Equimolar amount of the reaction INDIAN J. CHEM., SEC B, FEBRUARY 2020   232mixture was stirred at RT in the presence of piperidine as a catalyst. Compound (2) was prepared by the reaction of 3-acetyl-2H-chromen-2-one (1) and 2-cyanoacetohydrazide which was mixed in an equimolar amount in methanol (30 mL), glacial acetic acid and refluxed at 50-60°C. In the consequent step, intermediate (2) (0.01 mol) was heated with 2-(4-chlorobenzylidene)malononitrile (E) (0.01 mol).  In this reaction, piperidine was used as a catalyst  in ethanol (99%, 30 mL). As a result of this,  reaction mixture furnished compound (3). Compound (3) (0.01 mol) was taken in ethanol and further  reacted with aromatic aldehydes to produced novel compounds (4a-o).  Mechanism of the products 4a-o is proposed in the Scheme II. In the first step, hydrazone (A) undertook Michael addition with (B) and produced the intermediate (C). Intramolecular nucleophilic occurrence on the cyanide carbon was monitored  to yield intermediate (D). Compound (E)  was produced by the intramolecular electron  transfer to nitrogen atom. In the final step, intermediate (E) was altered to targeted molecules by intermolecular nucleophilic attack on carbonyl carbon of various aromatic aldehydes (Scheme II).  Discussion of Antimicrobial Activity  Antibacterial studies All the synthesized compounds 4a-o were screened for their in vitro activity against several panels of bacteria by the conventional broth micro-dilution method using Chloramphenicol as a standard drug and the results are presented in the Table I. It was observed that compounds 4g (-4-NO2), 4j (-4-Cl), 4k (-4-F) and 4o (-4-Br) were the most effective antibacterial agents. Compounds 4g (-4-NO2) and 4o (-4-Br) exhibited excellent activity against P. aeruginosa and S. aureus with 2 to 4 fold higher MIC (12.5-25 mg/mL) than chloramphenicol. Compounds 4j (-4-Cl) and 4k (-4-F) were the most effective against S. Pyogenes while compound 4j (-4-Cl) showed excellent activity against E. coli. Compounds 4g (-4-NO2) and 4o (-4-Br) have a very good activity against S. Pyogenes while compounds 4j (-4-Cl) and 4k (-4-F) were confirmed as very good agents against S. aureus. On the other hand, the presence of similar functional groups at the ortho position decrease the antibacterial activity as compared to 4e (-4-NO2) and 4h (-4-Cl). Remarkable antibacterial activity is depicted by groups like -NO2, -F, -Cl and -Br.  Antifungal studies Preliminary screening results showed that compounds 4d (-4-OH) and 4m (-4-OCH3) exhibited excellent activity against A. clavatus. While compound 4d (-4-OH) showed excellent activity against A. niger having 2 to 4 fold higher MIC (12.5-25 mg/mL) in comparison with a standard drug Nystatin. The enhancement of the activity of compounds 4d and 4m (-4-OCH3) may be attributed   Figure 1 — Commercially available drugs containing 2-pyridone nucleus DESAI et al.: COUMARIN LINKED 2-PYRIDONE HETEROCYCLES   233to the presence of electron releasing group at para position. When we installed hydroxy group at meta position in 4c (-3-OH), it possessed very good activity against A. niger and good activity against A. clavatus. Introduction of methoxy group in 4m (-4-OCH3) demonstrated very good potency against A. niger.  Cytotoxicity studies In vitro cytotoxicity of 4a-o was tested against human cervical cancer cell line (HeLa) by the MTT colorimetric assay31,32 and Table II displayed  IC50 values.  Cytotoxicity results exposed that the derivatives 4d, 4g, 4j, 4k, 4m and 4o having no toxicity at concentration level 100 µM (IC50 > 100 µM), while other compounds possessed moderate activity. It was confirmed that none of these tested compounds demonstrated any significant cytotoxic properties on HeLa cell lines. Above results indicated that none of   Scheme I — Synthetic route for the preparation of title compounds 4a-o INDIAN J. CHEM., SEC B, FEBRUARY 2020   234the compounds displayed cytotoxicity against 3T3 cell lines (IC50 > 100 µM).  Molecular docking Compounds 4b and 4e were used for docking poses representatives (Table III) as active molecules in transferase (1HNJ) of organism E. coli (strain K12). The beta-Ketoacyl-acyl carrier protein synthase III (FabH) is a condensing enzyme that plays central roles in fatty acid biosynthesis. This protein was downloaded from protein data bank (PDB). Binding of the ligands is shown in Figure 2, where, carbonyl    Scheme II — Plausible mechanistic pathway for the synthesis of compounds 4a-o  Table I — Antimicrobial screening results of compounds 4a-o Compd -R Minimum inhibitory concentration (MIC) for bacteria (µg/mL) Minimum inhibitory concentration (MIC) for fungi (µg/mL) E.c. P.a. S.a. S.p.  C.a. A.n. A.c. 4a -H 250 250 150 150 200 500 500 4b -2-OH 500 500 500 500 250 250 200 4c -3-OH 500 500 250 500 500 50 100 4d -4-OH 200 200 250 250 250 12.5 25 4e -2-NO2 500 250 500 250 500 >1000 >1000 4f -3-NO2 250 100 250 100 1000 1000 1000 4g -4-NO2 100 25 12.5 50 1000 500 500 4h -2-Cl 500 500 200 250 500 1000 1000 4i -3-Cl 200 250 250 200 250 250 250 4j -4-Cl 12.5 100 50 25 500 500 1000 4k -4-F 100 100 50 12.5 250 >1000 >1000 4l -3-OCH3 250 250 250 250 250 250 250 4m -4-OCH3 250 200 500 500 100 50 25 4n -3,4,5-(OCH3)3 500 500 250 500 200 500 500 4o -4-Br 100 25 25 50 1000 >1000 >1000 Chloramphenicol 50 50 50 50 − − − Nystatin − − − − 100 100 100 Escherichia coli (E.c.) MTCC-442; Pseudomonas aeruginosa (P.a.) MTCC-441; Staphylococcus aureus (S.a.) MTCC-96; Streptococcus pyogenes (S.p.) MTCC-443; Candida albicans (C.a.) MTCC-227; Aspergillus niger (A.n.) MTCC-282; Aspergillus clavatus (A.c.) MTCC-1323. DESAI et al.: COUMARIN LINKED 2-PYRIDONE HETEROCYCLES   235 >C=O has hydrogen bonding with TYR 639 and ARG 630, and TYR 639 have pi-pi interaction with coumarin ring.  Experimental Section  Preparation of 3-acetyl-2H-chromen-2-one, 1 33 and (E)2-(4-chlorobenzylidene)malononitrile 34 as per the literature method  Preparation of 2-cyano-N'-(2-(2-oxo-2H-chromen-3-yl)propylidene)acetohydrazide, 2 A mixture of 3-acetyl-2H-chromen-2-one 1 (0.01mol) and 2-cyanoacetohydrazide (0.01 mol) in methanol (30 mL) was refluxed for 14 h and then cooled down to RT. The separated crystals were filtered, air dried and recrystallized from ethanol (95%). Yield: 72%, m.p. 159°C. Anal. Calcd for C15H13N3O3: C, 63.60; H, 4.63; N, 14.83. Found: C, 63.63; H, 4.56; N, 14.76%.  Preparation of 6-amino-4-(4-chlorophenyl)-2-oxo-1-((1-(2-oxo-2H-chromen-3-yl)ethylidene)amino)-1,2-dihydropyridine-3,5-dicarbonitrile, 3 A mixture containing 2-cyano-N'-(2-(2-oxo-2H-chromen-yl)propylidene) acetohydrazide 2 (0.01 mol), (E)-2-(4-chlorobenzylidene)malononitrile (0.01 Table II — Levels of cytotoxicity prompted by selected compounds on HeLa cells Compd Cytotoxicity (IC50 µM) a HeLa b 4c >100 4d 97.56 4g >100 4j >100 4k 98.16 4m 96.88 4o >100 Doxorubicin 3.24 a IC50 is the concentration required to inhibit 50% of cell growth b HeLa human cervical cancer cell line  Table III — Docking score Compd Transferase (g Score) 4b −0.946 4e −0.831 4d −0.551 4g −0.475 4c −0.073 4l 0.509 4f 1.391 Chloroamphinicol −2.538 Organism: E. coli    Figure 2 — Molecular docking poses of 4b A (2D), B (3D). 4e A1 (2D), B1 (3D). INDIAN J. CHEM., SEC B, FEBRUARY 2020   236mol) and 2 drops of piperidine in ethanol (95%)  (30 mL) was refluxed for 22 h. The mixture was then cooled down to RT and diluted with a few drops of water. The crystals formed were filtered, air dried and recrystallized from ethanol. Yield: 68%, m.p. 190°C. Anal. Calcd for C24H14ClN5O3: C, 63.24; H, 3.10; N, 15.36. Found: C, 63.27; H, 3.04; N, 15.43%.  General procedure of preparation of 6-((benzylidene)amino)-4-(4-chlorophenyl)-2-oxo-1- ((1-(2-oxo-2H-chromen-3-yl)ethylidene)amino)-1,2-dihydropyridine-3,5-dicarbonitrile, 4a-o Compound 3 (0.01mol), benzaldehyde (0.01 mol) and ethanol (95%) (30 mL) were taken in a round bottom flask and refluxed for 12 h. Separated solid was filtered, dried and recrystallized from ethanol (95%). Yield: 73%. m.p. 157°C. Anal. Calcd for C31H18ClN5O3: C, 68.45; H, 3.34; N, 12.87. Found: C, 68.39; H, 3.39; N, 12.81%. 6-((Benzylidene)amino)-4-(4-chlorophenyl)-2-oxo-1-((1-(2-oxo-2H-chromen-3-yl)ethyli dene) amino)-1,2-dihydropyridine-3,5-dicarbonitrile, 4a: Yield: 73%. m.p. 157-161°C. IR (KBr): 2927 (C-H, CH3), 2209 (-C≡N stretching, nitrile group), 1731 (>C=O stretching, coumarin ring), 1564, 1597 (>C=N-, >C=C< stretching, aromatic ring), 1588, 1407 (C-H bending, -CH=N linkage), 1254 (C-O-C coumarin ring), 729 (C-Cl stretching) cm−1; 1H NMR (300 MHz, DMSO-d6): δ 9.18 (s, 1H, Ar-CH=N-), 8.91 (s, 1H, C4 proton of coumarin), 7.25-7.89 (m, 13H, Ar-H of coumarin ring and phenyl ring), 1.38 (s, 3H, -C(CH3)=N-); 13C NMR (100 MHz, DMSO-d6): δ 14.4, 114.5, 115.5, 115.9(2), 116.3, 118.2, 123.1, 125.1, 127.7, 128.5, 128.8 (2), 128.9 (2), 129.4 (2), 130.3 (2), 130.8, 131.2, 133.2 (2), 133.5, 153.1, 153.4, 153.6, 155.4, 159.6, 160.0, 163.8, 169.6; LCMS: m/z 543.11 (M+). Anal. Calcd for C31H18ClN5O3: C, 68.45; H, 3.34; N, 12.87. Found: C, 68.39; H, 3.39; N, 12.81%.  Conclusion We have concluded on the basis of the biological activity results that the titled compounds 4a-o depend on the different types of substituent pattern on phenyl ring. Many of the synthesized motifs (4g, 4j, 4k and 4o) having electron withdrawing atoms /groups such as halogen and nitro at para and meta positions showed potent antibacterial results while compounds 4d and 4m having electron donating groups at para position showed potent antifungal results. The para position was more favorable as per the antimicrobial activity data of the compounds. Compounds (4d, 4g, 4j, 4k, 4m and 4o) revealed notable cytotoxic action. This new class of novel coumarin linked 2-pyridone scaffolds can be optimized to get lead for further generation of antimicrobial agents. Therefore, the in silico studies and the biological activity can be specifically related with each other and optimization of potent molecules recognized in the present study can be taken out for further progress.  Supplementary Information Supplementary information is available in the website http://nopr.niscair.res.in/handle/123456789/60.  Acknowledgements Authors are thankful to the University Grants Commission, New Delhi and Department of Science and Technology, New Delhi for financial support under the NON-SAP and DST-FIST programs respectively. Mr. Krunalsinh A Jadeja is thankful to UGC, New Delhi for UGC-MRP Project Fellowship [UGC F. No.-43- 164/2014(SR)].  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Synthesis, characterization, molecular docking studies and biological activity of coumarin linked 2-pyridone heterocycles

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Synthesis, characterization, molecular docking studies and biological activity of coumarin linked 2-pyridone heterocycles

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