A series of novel C4-C7-tethered biscoumarin derivatives (12a-e) linked through piperazine moiety was designed, synthesized, and evaluated biological/therapeutic potential. Biscoumarin 12d was found to be the most effective inhibitor of both acetylcholinesterase (AChE, IC50 = 6.30 µM) and butyrylcholinesterase (BChE, IC50 = 49 µM). Detailed molecular modelling studies compared the accommodation of ensaculin (well-established coumarin derivative tested in phase I of clinical trials) and 12d in the human recombinant AChE (hAChE) active site. The ability of novel compounds to cross the blood-brain barrier (BBB) was predicted with a positive outcome for compound 12e. The antiproliferative effects of newly synthesized biscoumarin derivatives were tested in vitro on human lung carcinoma cell line (A549) and normal colon fibroblast cell line (CCD-18Co). The effect of derivatives on cell proliferation was evaluated by MTT assay, quantification of cell numbers and viability, colony-forming assay, analysis of cell cycle distribution and mitotic activity. Intracellular localization of used derivatives in A549 cells was confirmed by confocal microscopy. Derivatives 12d and 12e showed significant antiproliferative activity in A549 cancer cells without a significant effect on normal CCD-18Co cells. The inhibition of hAChE/human recombinant BChE (hBChE), the antiproliferative activity on cancer cells, and the ability to cross the BBB suggest the high potential of biscoumarin derivatives. Beside the treatment of cancer, 12e might be applicable against disorders such as schizophrenia, and 12d could serve future development as therapeutic agents in the prevention and/or treatment of Alzheimer's disease.

Biomedical Research Centre University Hospital Hradec Kralove Sokolska 581 500 05 Hradec Kralove Czech Republic

Department of Biochemistry Faculty of Science Pavol Jozef Šafárik University in Košice Šrobárova 2 041 54 Košice Slovakia

Department of Cellular Biology Faculty of Science Pavol Jozef Šafárik University in Košice Šrobárova 2 041 54 Košice Slovakia

Department of Organic Chemistry Institute of Chemical Sciences Faculty of Science Pavol Jozef Šafárik University in Košice Moyzesova 11 040 01 Kosice Slovakia

Department of Toxicology and Military Pharmacy Faculty of Military Health Sciences University of Defense Trebesska 1575 500 05 Hradec Kralove Czech Republic

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Flemingovo Náměstí 542 2 160 00 Prague 6 Czech Republic

National Institute of Mental Health Topolová 748 250 67 Klecany Czech Republic

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Peng X.-M., Damu G.L.V., Zhou C.-H. Current developments of coumarin compounds in medicinal chemistry. Curr. Pharm. Des. 2013;19:3884–3930. doi: 10.2174/1381612811319210013. PubMed DOI

Gómez-Outes A., Suárez-Gea M.L., Calvo-Rojas G., Lecumberri R., Rocha E., Pozo-Hernández C., Terleira-Fernández A.I., Vargas-Castrillón E. Discovery of anticoagulant drugs: A historical perspective. Curr. Drug Discov. Technol. 2012;9:83–104. doi: 10.2174/1570163811209020083. PubMed DOI

Anand P., Singh B., Singh N. A Review on coumarins as acetylcholinesterase inhibitors for Alzheimer’s disease. Bioorg. Med. Chem. 2012;20:1175–1180. doi: 10.1016/j.bmc.2011.12.042. PubMed DOI

Kostova I., Bhatia S., Grigorov P., Balkansky S., Parmar V.S., Prasad A.K., Saso L. Coumarins as antioxidants. Curr. Med. Chem. 2011;18:3929–3951. doi: 10.2174/092986711803414395. PubMed DOI

Riveiro M.E., De Kimpe N., Moglioni A., Vázquez R., Monczor F., Shayo C., Davio C. Coumarins: Old compounds with novel promising therapeutic perspectives. Curr. Med. Chem. 2010;17:1325–1338. doi: 10.2174/092986710790936284. PubMed DOI

Wu L., Wang X., Xu W., Farzaneh F., Xu R. The structure and pharmacological functions of coumarins and their derivatives. Curr. Med. Chem. 2009;16:4236–4260. doi: 10.2174/092986709789578187. PubMed DOI

Cravotto G., Nano G.M., Palmisano S.G., Tagliapietra S. The Chemistry of Coumarin Derivatives, Part XIII. The Reactivity of 4-Hydroxycoumarin under Heterogenous High Intensity Sonochemical Conditions. Synthesis. 2003;34:1286–1291.

Shabbir M., Sultani S.Z., Jabbar A., Choudhary M.I. Cinnamates and coumarins from the leaves of Murraya paniculata. Phytochemistry. 1997;44:683–685.

Al-Amiery A.A., Al-Bayati R.I.H., Saour K.Y., Radi M.F. Cytotoxicity, antioxidant, and antimicrobial activities of novel 2-quinolone derivatives derived from coumarin. Res. Chem. Intermed. 2012;38:559–569. doi: 10.1007/s11164-011-0371-2. DOI

Negi N., Ochi A., Kurosawa M., Ushijima K., Kitaguchi Y., Kusakabe E., Okasho F., Kimachi T., Teshima N., Ju-Ichi M., et al. Two new dimeric coumarins isolated from Murraya exotica. Chem. Pharm. Bull. 2005;53:1180–1182. doi: 10.1248/cpb.53.1180. PubMed DOI

Tan G., Yao Y., Gu Y., Li S., Lv M., Wang K., Chen H., Li X. Cytotoxicity and DNA binding property of the dimers of triphenylethylene- coumarin hybrid with one amino side chain. Bioorganic Med. Chem. Lett. 2014;24:2825–2830. doi: 10.1016/j.bmcl.2014.04.106. PubMed DOI

Kurt B.Z., Dag A., Doğan B., Durdagi S., Angeli A., Nocentini A., Supuran C.T., Sonmez F. Synthesis, biological activity and multiscale molecular modeling studies of bis-coumarins as selective carbonic anhydrase IX and XII inhibitors with effective cytotoxicity against hepatocellular carcinoma. Bioorg. Chem. 2019;87:838–850. doi: 10.1016/j.bioorg.2019.03.003. PubMed DOI

Umar M.I., Saeed A., Ejaz S.A., Sévigny J., Iqbal J., Ibrar A., Lecka J. Expanding the alkaline phosphatase inhibition, cytotoxic and proapoptotic profile of biscoumarin-iminothiazole and coumarin-triazolothiadiazine conjugates. ChemistrySelect. 2018;3:13377–13386.

Morsy S.A., Farahat A.A., Nasr M.N.A., Tantawy A.S. Synthesis, Molecular modeling and anticancer activity of new coumarin containing compounds. Saudi Pharm. J. 2017;25:873–883. doi: 10.1016/j.jsps.2017.02.003. PubMed DOI PMC

Xu J., Ai J., Liu S., Peng X., Yu L., Geng M., Nan F. Design and synthesis of 3,3′-biscoumarin-based c-Met inhibitors. Org. Biomol. Chem. 2014;12:3721–3734. doi: 10.1039/C4OB00364K. PubMed DOI

Xie S.S., Wang X., Jiang N., Yu W., Wang K.D.G., Lan J.S., Li Z.R., Kong L.Y. Multi-target tacrine-coumarin hybrids: Cholinesterase and monoamine oxidase b inhibition properties against Alzheimer’s disease. Eur. J. Med. Chem. 2015;95:153–165. doi: 10.1016/j.ejmech.2015.03.040. PubMed DOI

Joubert J., Foka G.B., Repsold B.P., Oliver D.W., Kapp E., Malan S.F. Synthesis and evaluation of 7-substituted coumarin derivatives as multimodal monoamine oxidase-B and cholinesterase inhibitors for the treatment of Alzheimer’s disease. Eur. J. Med. Chem. 2017;125:853–864. doi: 10.1016/j.ejmech.2016.09.041. PubMed DOI

Hilgert M., Nöldner M., Chatterjee S.S., Klein J. KA-672 Inhibits rat brain acetylcholinesterase in vitro but not in vivo. Neurosci. Lett. 1999;263:193–196. doi: 10.1016/S0304-3940(99)00149-4. PubMed DOI

Borumandnia N. Worldwide Patterns in Alzheimer’s Disease and Other Dementias Prevalence from 1990 to 2017: A Growth Mixture Models Approach Analysis of Trends. Res. Sq. 2020 doi: 10.21203/rs.3.rs-25512/v1. DOI

Guo J., Cheng J., North B.J., Wei W. Functional analyses of major cancer-related signaling pathways in Alzheimer’s disease etiology. Biochim. Biophys. Acta Rev. Cancer. 2017;1868:341–358. doi: 10.1016/j.bbcan.2017.07.001. PubMed DOI PMC

Majd S., Power J., Majd Z. Alzheimer’s disease and cancer: When two monsters cannot be together. Front. Neurosci. 2019;13:155. doi: 10.3389/fnins.2019.00155. PubMed DOI PMC

Monacelli F., Cea M., Borghi R., Odetti P., Nencioni A. Do cancer drugs counteract neurodegeneration? Repurposing for Alzheimer’s disease. J. Alzheimer’s Dis. 2017;55:1295–1306. doi: 10.3233/JAD-160840. PubMed DOI

Mehta M., Adem A., Sabbagh M. New acetylcholinesterase inhibitors for alzheimer’s disease. Int. J. Alzheimer’s Dis. 2012;2012:8. doi: 10.1155/2012/728983. PubMed DOI PMC

Piazzi L., Cavalli A., Colizzi F., Belluti F., Bartolini M., Mancini F., Recanatini M., Andrisano V., Rampa A. Multi-target-directed coumarin derivatives: hAChE and BACE1 inhibitors as potential anti-alzheimer compounds. Bioorg. Med. Chem. Lett. 2008;18:423–426. doi: 10.1016/j.bmcl.2007.09.100. PubMed DOI

Stefanachi A., Leonetti F., Pisani L., Catto M., Carotti A. Coumarin: A natural, privileged and versatile scaffold for bioactive compounds. Molecules. 2018;23:250. doi: 10.3390/molecules23020250. PubMed DOI PMC

Kancheva V.D., Boranova P.V., Nechev J.T., Manolov I.I. Structure-activity relationships of new 4-hydroxy bis-coumarins as radical scavengers and chain-breaking antioxidants. Biochimie. 2010;92:1138–1146. doi: 10.1016/j.biochi.2010.02.033. PubMed DOI

Chimenti F., Secci D., Bolasco A., Chimenti P., Granese A., Carradori S., Befani O., Turini P., Alcaro S., Ortuso F. Synthesis, Molecular modeling studies, and selective inhibitory activity against monoamine oxidase of N,N’-Bis[2 -oxo-2H-benzopyran]-3-carboxamides. Bioorg. Med. Chem. Lett. 2006;16:4135–4140. doi: 10.1016/j.bmcl.2006.04.026. PubMed DOI

Barta J.A., Powell C.A., Wisnivesky J.P. Global epidemiology of lung cancer. Ann. Glob. Health. 2019;85:8. doi: 10.5334/aogh.2419. PubMed DOI PMC

Xi H.J., Wu R.P., Liu J.J., Zhang L.J., Li Z.S. Role of acetylcholinesterase in lung cancer. Thorac. Cancer. 2015;6:390–398. doi: 10.1111/1759-7714.12249. PubMed DOI PMC

Friedman J.R., Richbart S.D., Merritt J.C., Brown K.C., Nolan N.A., Akers A.T., Lau J.K., Robateau Z.R., Miles S.L., Dasgupta P. Acetylcholine Signaling System in Progression of Lung Cancers. Pharmacol. Ther. 2019;194:222–254. doi: 10.1016/j.pharmthera.2018.10.002. PubMed DOI PMC

Vidal C.J. Expression of cholinesterases in brain and non-brain tumours. Chem. Biol. Interact. 2005;157–158:227–232. doi: 10.1016/j.cbi.2005.10.035. PubMed DOI

Ballard C.G., Greig N.H., Guillozet-Bongaarts A.L., Enz A., Darvesh S. Cholinesterases: Roles in the brain during health and disease. Curr. Alzheimer Res. 2005;2:307–318. doi: 10.2174/1567205054367838. PubMed DOI

Johnson G., Moore S.W. The Adhesion Function on Acetylcholinesterase Is Located at the Peripheral Anionic Site. Biochem. Biophys. Res. Commun. 1999;258:758–762. doi: 10.1006/bbrc.1999.0705. PubMed DOI

Pérez-Aguilar B., Vidal C.J., Palomec G., García-Dolores F., Gutiérrez-Ruiz M.C., Bucio L., Gómez-Olivares J.L., Gómez-Quiroz L.E. Acetylcholinesterase is associated with a decrease in cell proliferation of hepatocellular carcinoma cells. Biochim. Biophys. Acta Mol. Basis Dis. 2015;1852:1380–1387. doi: 10.1016/j.bbadis.2015.04.003. PubMed DOI

Price D., Muterspaugh R., Clegg B., Williams A., Stephens A., Guthrie J., Heyl D., Evans H.G. IGFBP-3 blocks hyaluronan-CD44 signaling, leading to increased acetylcholinesterase levels in A549 cell media and apoptosis in a p53-dependent manner. Sci. Rep. 2020;10:5083. doi: 10.1038/s41598-020-61743-3. PubMed DOI PMC

Campoy F.J., Vidal C.J., Muñoz-Delgado E., Montenegro M.F., Cabezas-Herrera J., Nieto-Cerón S. Cholinergic system and cell proliferation. Chem. Biol. Interact. 2016;259:257–265. doi: 10.1016/j.cbi.2016.04.014. PubMed DOI

Zhang X.J., Greenberg D.S. Acetylcholinesterase involvement in apoptosis. Front. Mol. Neurosci. 2012;5:40. doi: 10.3389/fnmol.2012.00040. PubMed DOI PMC

Lu L., Zhang X., Zhang B., Wu J., Zhang X. Synaptic acetylcholinesterase targeted by MicroRNA-212 functions as a tumor suppressor in non-small cell lung cancer. Int. J. Biochem. Cell Biol. 2013;45:2530–2540. doi: 10.1016/j.biocel.2013.08.007. PubMed DOI

Calaf G.M., Parra E., Garrido F. Cell proliferation and tumor formation induced by Eserine, an acetylcholinesterase inhibitor, in rat mammary gland. Oncol. Rep. 2007;17:25–33. doi: 10.3892/or.17.1.25. PubMed DOI

Chen X.-M., Zhang Z.-J., Liu A.-L., LV F.-J., LI S.-F. Cholinesterase and human lung cancer cells (A-549) inhibitory activity of the Cassava Peel of Euphorbiaceae in vitro; Proceedings of the DEStech Transactions on Environment, Energy and Earth Sciences; Joint International Conference on Social Science and Environmental Science (SSES 2016) and International Conference on Food Science and Engineering (ICFSE 2016); Guangzhou, China. 15–16 October 2016; pp. 389–394.

Nguyen T.-H.-T., Pham H.-V.-T., Pham N.-K.-T., Quach N.-D.-P., Pudhom K., Hansen P.E., Nguyen K.-P.-P. Chemical constituents from Sonneratia ovata backer and their in vitro cytotoxicity and acetylcholinesterase inhibitory activities. Bioorg. Med. Chem. Lett. 2015;25:2366–2371. doi: 10.1016/j.bmcl.2015.04.017. PubMed DOI

Zovko A., Sepcic K., Turk T., Faimali M., Garaventa F., Chelossi E., Paleari L., Falugi C., Aluigi M., Angelini C., et al. New aspects of the relationship between acetylcholinesterase activity and cancer I: Poly-Aps experiments. WSEAS Trans. Biol. Biomed. 2009;6:58–69.

Lee J., Sohn E.J., Yoon S.W., Kim C.G., Lee S., Kim J.Y., Baek N., Kim S.-H. Anti-metastatic effect of dehydrocorydaline on H1299 non-small cell lung carcinoma cells via inhibition of matrix metalloproteinases and B cell lymphoma 2. Phyther. Res. 2017;31:441–448. doi: 10.1002/ptr.5766. PubMed DOI

Deng R., Li W., Guan Z., Zhou J.-M., Wang Y., Mei Y.-P., Li M.-T., Feng G.-K., Huang W., Liu Z.-C., et al. Acetylcholinesterase expression mediated by c-Jun-NH2-terminal kinase pathway during anticancer drug-induced apoptosis. Oncogene. 2006;25:7070–7077. doi: 10.1038/sj.onc.1209686. PubMed DOI

Syed M., Fenoglio-Preiser C., Skau K.A., Weber G.F. Acetylcholinesterase supports anchorage independence in colon cancer. Clin. Exp. Metastasis. 2008;25:787–798. doi: 10.1007/s10585-008-9192-0. PubMed DOI

Arafath M.A., Adam F., Al-Suede F.S.R., Razali M.R., Ahamed M.B.K., Abdul Majid A.M.S., Hassan M.Z., Osman H., Abubakar S. Synthesis, Characterization, X-ray crystal structures of heterocyclic schiff base compounds and in vitro cholinesterase inhibition and anticancer activity. J. Mol. Struct. 2017;1149:216–228. doi: 10.1016/j.molstruc.2017.07.092. DOI

Ozmen Ozgun D., Gul H.I., Yamali C., Sakagami H., Gulcin I., Sukuroglu M., Supuran C.T. Synthesis and bioactivities of pyrazoline benzensulfonamides as carbonic anhydrase and acetylcholinesterase inhibitors with low cytotoxicity. Bioorg. Chem. 2019;84:511–517. doi: 10.1016/j.bioorg.2018.12.028. PubMed DOI

Xu J., Zhao S., Zhang S., Pei J., Li Y., Zhang Y., He X., Hu L. Development of a multivalent acetylcholinesterase inhibitor via dynamic combinatorial chemistry. Int. J. Biol. Macromol. 2020;150:1184–1191. doi: 10.1016/j.ijbiomac.2019.10.127. PubMed DOI

Laskowski S.C., Clinton R.O. Coumarins. II. Derivatives of coumarin-3- and -4-acetic acids. J. Am. Chem. Soc. 1950;72:3987–3991. doi: 10.1021/ja01165a043. DOI

Di L., Kerns E.H., Fan K., McConnell O.J., Carter G.T. High throughput artificial membrane permeability assay for blood-brain barrier. Eur. J. Med. Chem. 2003;38:223–232. doi: 10.1016/S0223-5234(03)00012-6. PubMed DOI

Carpenter T.S., Kirshner D.A., Lau E.Y., Wong S.E., Nilmeier J.P., Lightstone F.C. A Method to predict blood-brain barrier permeability of drug-like compounds using molecular dynamics simulations. Biophys. J. 2014;107:630–641. doi: 10.1016/j.bpj.2014.06.024. PubMed DOI PMC

Cheung J., Rudolph M.J., Burshteyn F., Cassidy M.S., Gary E.N., Love J., Franklin M.C., Height J.J. Structures of human acetylcholinesterase in complex with pharmacologically important ligands. J. Med. Chem. 2012;55:10282–10286. doi: 10.1021/jm300871x. PubMed DOI

Hoerr R., Noeldner M. Ensaculin (KA-672 HCl): A multitransmitter approach to dementia treatment. CNS Drug Rev. 2002;8:143–158. doi: 10.1111/j.1527-3458.2002.tb00220.x. PubMed DOI PMC

Lacy A., O’Kennedy R. Studies on coumarins and coumarin-related compounds to determine their therapeutic role in the treatment of cancer. Curr. Pharm. Des. 2005;10:3797–3811. doi: 10.2174/1381612043382693. PubMed DOI

Huang R.-Z., Hua S.-X., Wang C.-Y., Pan Y.-M., Qin J.-M., Ding Z.-Y., Zhang Y., Wang H.-S. 4-Methylumbelliferones analogues as anticancer agents: Synthesis and in cell pharmacological studies. Anticancer. Agents Med. Chem. 2017;17:576–589. doi: 10.2174/1871520616666160926113109. PubMed DOI

Leevy W.M., Weber M.E., Gokel M.R., Hughes-Strange G.B., Daranciang D.D., Ferdani R., Gokel G.W. Correlation of bilayer membrane cation transport and biological activity in alkyl-substituted lariat ethers. Org. Biomol. Chem. 2005;3:1647–1652. doi: 10.1039/b418194h. PubMed DOI PMC

Supek F., Ramljak T.Š., Marjanović M., Buljubašić M., Kragol G., Ilić N., Šmuc T., Zahradka D., Mlinarić-Majerski K., Kralj M. Could LogP be a principal determinant of biological activity in 18-crown-6 ethers? Synthesis of biologically active adamantane-substituted diaza-crowns. Eur. J. Med. Chem. 2011;46:3444–3454. doi: 10.1016/j.ejmech.2011.05.009. PubMed DOI

Bortner C.D., Cidlowski J.A. Cell shrinkage and monovalent cation fluxes: Role in apoptosis. Arch. Biochem. Biophys. 2007;462:176–188. doi: 10.1016/j.abb.2007.01.020. PubMed DOI PMC

Bisi A., Cappadone C., Rampa A., Farruggia G., Sargenti A., Belluti F., Di Martino R.M.C., Malucelli E., Meluzzi A., Iotti S., et al. Coumarin derivatives as potential antitumor agents: Growth inhibition, apoptosis induction and multidrug resistance reverting activity. Eur. J. Med. Chem. 2017;127:577–585. doi: 10.1016/j.ejmech.2017.01.020. PubMed DOI

Zhang L., Mizumoto K., Sato N., Ogawa T., Kusumoto M., Niiyama H., Tanaka M. Quantitative determination of apoptotic death in cultured human pancreatic cancer cells by propidium iodide and digitonin. Cancer Lett. 1999;142:129–137. doi: 10.1016/S0304-3835(99)00107-X. PubMed DOI

Firmino S.S., André S.C., Hastenreiter Z., Campos V.K. In vitro assessment of the cytotoxicity of Gallium (III) complexes with Isoniazid-Derived Hydrazones: Effects on clonogenic survival of HCT-116 cells. Inorganica Chim. Acta. 2019;497:119079. doi: 10.1016/j.ica.2019.119079. DOI

Pettersen E.F., Goddard T.D., Huang C.C., Couch G.S., Greenblatt D.M., Meng E.C., Ferrin T.E. UCSF Chimera—A Visualization system for exploratory research and analysis. J. Comput. Chem. 2004;25:1605–1612. doi: 10.1002/jcc.20084. PubMed DOI

Morris G.M., Huey R., Lindstrom W., Sanner M.F., Belew R.K., Goodsell D.S., Olson A.J. AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J. Comput. Chem. 2009;30:2785–2791. doi: 10.1002/jcc.21256. PubMed DOI PMC

Panek D., Więckowska A., Wichur T., Bajda M., Godyń J., Jończyk J., Mika K., Janockova J., Soukup O., Knez D., et al. Design, synthesis and biological evaluation of new phthalimide and saccharin derivatives with alicyclic amines targeting cholinesterases, beta-secretase and amyloid beta aggregation. Eur. J. Med. Chem. 2017;125:676–695. doi: 10.1016/j.ejmech.2016.09.078. PubMed DOI

Hepnarova V., Korabecny J., Matouskova L., Jost P., Muckova L., Hrabinova M., Vykoukalova N., Kerhartova M., Kucera T., Dolezal R., et al. The concept of hybrid molecules of tacrine and benzyl quinolone carboxylic acid (BQCA) as multifunctional agents for Alzheimer’s disease. Eur. J. Med. Chem. 2018;150:292–306. doi: 10.1016/j.ejmech.2018.02.083. PubMed DOI

Svobodova B., Mezeiova E., Hepnarova V., Hrabinova M., Muckova L., Kobrlova T., Jun D., Soukup O., Jimeno M.L., Marco-Contelles J., et al. Exploring structure-activity relationship in tacrine-squaramide derivatives as potent cholinesterase inhibitors. Biomolecules. 2019;9:379. doi: 10.3390/biom9080379. PubMed DOI PMC

O’Boyle N.M., Banck M., James C.A., Morley C., Vandermeersch T., Hutchison G.R. Open Babel: An open chemical toolbox. J. Cheminform. 2011;3:33. doi: 10.1186/1758-2946-3-33. PubMed DOI PMC

Trott O., Olson A.J. AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 2010;31:455–461. doi: 10.1002/jcc.21334. PubMed DOI PMC