Synthesis and Evaluation of 1,4-Dihydropyridine-Based Urea Derivatives as Polyphenol Oxidase Inhibitors

Öz

This study investigated the potential inhibitory effects of nine novel synthesized urea-substituted 1,4-dihydropyridine derivatives (DT-DEN-1-9) on polyphenol oxidase (PPO) activity. The compounds were synthesized via the Hantzsch reaction, providing a series of structurally diverse urea and thiourea-modified 1,4-dihydropyridines. Polyphenol oxidase enzyme was extracted from banana (Musa cavendishii) and purified using affinity chromatography with a Sepharose 4B-L-tyrosine-p-aminobenzoic acid affinity gel. The purified enzyme's activity was measured spectrophotometrically using catechol as the substrate, monitoring the increase in absorbance at 420 nm. The inhibitory effects of the synthesized compounds on PPO activity were evaluated through in vitro assays. Various concentrations of each compound were incorporated into the enzyme reaction mixture, and the residual PPO activity was determined. The percentage of PPO activity was calculated relative to a control reaction without inhibitors. IC50 values, representing the concentration of inhibitor required to reduce enzyme activity by 50%, were determined using Lineweaver-Burk plots. Among the tested compounds, DT-DEN-6, featuring a phenyl thiourea substituent, exhibited the most potent inhibition with an IC50 value of 100.14 μM. DT-DEN-8, containing a 2,5-dichlorophenyl thiourea moiety, also showed strong inhibitory activity with an IC50 below 150 μM. Structure-activity relationships were observed, with electron-withdrawing substituents generally enhancing inhibitory potency. Conversely, DT-DEN-5, bearing a 4-(trifluoromethyl)phenyl thiourea substituent, exhibited the weakest inhibition profile (IC50: 233.33 μM). Our findings provide valuable insights for the design of next-generation PPO inhibitors, potentially leading to the development of novel anti-browning agents for applications in food preservation and other industries where control of enzymatic browning is crucial.

Anahtar Kelimeler

• 1,4-Dihydropyridine
• urea derivatives
• polyphenol oxidase
• inhibitory effect
• Hantzsch reaction
• prediction of activity spectra for substances

Kaynakça

1. Arslan, O., Erzengin, M., Sinan, S., Ozensoy, O., 2004. Purification of mulberry (Morus alba L.) polyphenol oxidase by affinity chromatography and investigation of its kinetic and electrophoretic properties. Food Chemistry, 88(3): 479-484.
2. Batista, K.A., Batista, G.L., Alves, G.L., Fernandes, K.F., 2014. Extraction, partial purification and characterization of polyphenol oxidase from Solanum lycocarpum fruits. Journal of Molecular Catalysis B: Enzymatic, 102: 211-217.
3. Ben-Shalom, N., Kahn, V., Harel, E., Mayer, A.M., 1977. Catechol oxidase from green olives: Properties and partial purification. Phytochemistry, 16(8): 1153-1158.
4. Bryzgalov, A.O., Dolgikh, M.P., Sorokina, I.V., Tolstikova, T.G., Sedova, V.F., Shkurko, O.P., 2006. Antiarrhythmic activity of 4, 6-di (het) aryl-5-nitro-3, 4-dihydropyrimidin-(1H)-2-ones and its effects on arterial pressure in rats. Bioorganic & Medicinal Chemistry Letters, 16(5): 1418-1420.
5. Chilaka, F.C., Eze, S., Anyadiegwu, C., Uvere, P.O., 2002. Browning in processed yams: Peroxidase or polyphenol oxidase? Journal of the Science of Food and Agriculture, 82(8): 899-903.
6. Cho, S.J., Roh, J.S., Sun, W.S., Kim, S.H., Park, K.D., 2006. N-Benzylbenzamides: A new class of potent tyrosinase inhibitors. Bioorganic & Medicinal Chemistry Letters, 16(10): 2682-2684.
7. Datar, P.A., Auti, P.B., 2012. 2DQSAR of novel 4-substituted 1, 4 dihydropyridines-3, 5-dicarboxylate as potential antihypertensive agent. Journal of Computational Methods in Molecular Design, 2(3): 85-91.
8. Demirci, T., Çelik, B., Yıldız, Y., Eriş, S., Arslan, M., Sen, F., Kilbas, B., 2016. Retracted article: One-pot synthesis of Hantzsch dihydropyridines using a highly efficient and stable PdRuNi@ GO catalyst. RSC Advances, 6(80): 76948-76956.

9. Diwakar, S.K., Naik, G., Mishra, S.K., 2015a. Polyphenol oxidase enzyme: A review. In: A.K. Dewivedi (Ed.), Transdisciplinary Environmental Issues, 1st Edn., AV Academikerverlag GmbH & Co. KG., Germany, pp. 39-88.
10. Diwakar, S.T., Radhakrishnan-Vinod, K., Chanda, S.V., 2015b. Molecular characterization of Aspergillus flavus isolatesfrom peanut fields in India using AFLP. Brazilian Journal of Microbiology, 46(3): 673-682.
11. Erzengin, M., 2009. Affinity purification and characterization of polyphenol oxidase from Helianthus tuberosus L. Hacettepe Journal of Biology and Chemistry, 37(4): 313-325.
12. Filimonov, D.A., Lagunin, A.A., Gloriozova, T.A., Rudik, A.V., Druzhilovskii, D.S., Pogodin, P.V., Poroikov, V.V., 2014. Prediction of the biological activity spectra of organic compounds using the PASS online web resource. Chemistry of Heterocyclic Compounds, 50(3): 444-457.
13. Huang, X., Su, J., Rao, A.U., Tang, H., Zhou, W., Zhu, X., Chen, X., Liu, Z., Huang, Y., Degrado, S., Xiao, D., Qin, J., Aslanian, R., McKittrick, B.A., Greenfeder, S., Heek, M.V, Chintala, M., Palani, A., 2012. SAR studies of C2 ethers of 2H-pyrano [2, 3-d] pyrimidine-2, 4, 7 (1H, 3H)-triones as nicotinic acid receptor (NAR) agonist. Bioorganic & Medicinal Chemistry Letters, 22(2): 854-858.
14. Kamal, A., Gasmalla, M.A., Alyousef, H., 2015. Efficient methods for polyphenol oxidase production. International Journal of Nutrition and Food Sciences, 4(6): 656-659.
15. Kaya, M.O., Demirci, T., Çalışır, Ü., Özdemir, O., Kaya, Y., Arslan, M., 2024. Synthesis, activatory effects, molecular docking and ADME studies as rabbit muscle pyruvate kinase activators of ureido phenyl substituted 1, 4-dihydropyridine derivatives. Research on Chemical Intermediates, 50(1): 437-463.
16. Khoshneviszadeh, M., Edraki, N., Javidnia, K., Alborzi, A., Pourabbas, B., Mardaneh, J., Miri, R., 2009. Synthesis and biological evaluation of some new 1, 4-dihydropyridines containing different ester substitute and diethyl carbamoyl group as anti-tubercular agents. Bioorganic & Medicinal Chemistry, 17(4): 1579-1586.
17. Lacki, K., Duvnjak, Z., 1999. Stability of a polyphenol oxidase from the white‐rot fungus trametes versicolor in the presence of canola meal. Acta Biotechnologica, 19(2): 91-100.
18. Lineweaver, H., Burk, D., 1934. The determination of enzyme dissociation constants. Journal of the American Chemical Society, 56(3): 658-666.
19. Marín-Prida, J., Pardo Andreu, G.L., Rossignoli, C.P., Durruthy, M.G., Rodríguez, E.O., Reyes, Y.V., Acosta, R.F., Uyemura, S.A., Alberici, L.C., 2017. The cytotoxic effects of VE-3N, a novel 1, 4-dihydropyridine derivative, involve the mitochondrial bioenergetic disruption via uncoupling mechanisms. Toxicology in Vitro, 42: 21-30.
20. Mayer, A.M., 2006. Polyphenol oxidases in plants and fungi: Going places? A review. Phytochemistry, 67(21): 2318-2331.
21. Mishra, B.B., Gautam, S., 2016. Polyphenol oxidases: Biochemical and molecular characterization, distribution, role and its control. Enzyme Engineering, 5(1): 141-149.
22. Nava-Zuazo, C., Estrada-Soto, S., Guerrero-Álvarez, J., León-Rivera, I., Molina-Salinas, G.M., Said-Fernández, S., Chan-Bacab, M.J., Cedillo-Rivera, R., Moo-Puc, R., Mirón-López, G., Navarrete-Vazquez, G., 2010. Design, synthesis, and in vitro antiprotozoal, antimycobacterial activities of N-{2-[(7-chloroquinolin-4-yl)amino]ethyl}ureas. Bioorganic & Medicinal Chemistry, 18(17): 6398-6403.
23. Pedemonte, N., Boido, D., Moran, O., Giampieri, M., Mazzei, M., Ravazzolo, R., Galietta, L.J., 2007. Structure-activity relationship of 1, 4-dihydropyridines as potentiators of the cystic fibrosis transmembrane conductance regulator chloride channel. Molecular Pharmacology, 72(1): 197-207.
24. Queiroz, C., Mendes Lopes, M.L., Fialho, E., Valente-Mesquita, V.L., 2008. Polyphenol oxidase: Characteristics and mechanisms of browning control. Food Reviews International, 24(4): 361-375.
25. Stout, D.M., Meyers, A.I., 1982. Recent advances in the chemistry of dihydropyridines. Chemical Reviews, 82(2): 223-243.
26. Tale, R.H., Rodge, A.H., Hatnapure, G.D., Keche, A.P., Patil, K.M., Pawar, R.P., 2013. The synthesis, anti-inflammatory, and anti-microbial activity evaluation of new series of 4-(3-arylureido) phenyl-1, 4-dihydropyridine urea derivatives. Medicinal Chemistry Research, 22(3): 1450-1455.
27. Teleb, M., Zhang, F.X., Huang, J., Gadotti, V.M., Farghaly, A.M., AboulWafa, O.M., Zamponi, G.W., Fahmy, H., 2017. Synthesis and biological evaluation of novel N3-substituted dihydropyrimidine derivatives as T-type calcium channel blockers and their efficacy as analgesics in mouse models of inflammatory pain. Bioorganic & Medicinal Chemistry, 25(6): 1926-1938.
28. Triggle, D.J., 2003. 1, 4-Dihydropyridines as calcium channel ligands and privileged structures. Cellular and Molecular Neurobiology, 23(3): 293-303.
29. Trivedi, A., Dodiya, D., Dholariya, B., Kataria, V., Bhuva, V., Shah, V., 2011. Synthesis and biological evaluation of some novel 1,4‐dihydropyridines as potential antitubercular agents. Chemical Biology & Drug Design, 78(5): 881-886.
30. Ulloora, S., Shabaraya, R., Ranganathan, R., Adhikari, A.V., 2013. Synthesis, anticonvulsant and anti-inflammatory studies of new 1,4-dihydropyridin-4-yl-phenoxyacetohydrazones. European Journal of Medicinal Chemistry, 70: 341-349.
31. Vaidya, B.K., Suthar, H.K., Kasture, S., Nene, S., 2006. Purification of potato polyphenol oxidase (PPO) by partitioning in aqueous two-phase system. Biochemical Engineering Journal, 28(2): 161-166.
32. Viveka, S., Dinesha, Madhu, L.N., Nagaraja, G.K., 2015. Synthesis of new pyrazole derivatives via multicomponent reaction and evaluation of their antimicrobial and antioxidant activities. Monatshefte für Chemie-Chemical Monthly, 146(9): 1547-1555.
33. Wesche‐Ebeling, P.E.D.R.O., Montgomery, M.W., 1990. Strawberry polyphenoloxidase: Extraction and partial characterization. Journal of Food Science, 55(5): 1320-1324.
34. Ziarani, G.M., Gholamzadeh, P., Badiei, A., Asadi, S., Soorki, A.A., 2015. Application of SBA-15 functionalized sulfonic acid (SBA-Pr-SO3H) as an efficient nanoreactor in the one- pot synthesis of pyrido [2, 3-d] pyrimidine. Journal of the Chilean Chemical Society, 60(2): 2975-2978.