Therapeutic options for Alzheimer's disease are limited. Dual compounds targeting two pathways concurrently may enable enhanced effect. The study focuses on tacrine derivatives inhibiting acetylcholinesterase (AChE) and simultaneously N-methyl-D-aspartate (NMDA) receptors. Compounds with balanced inhibitory potencies for the target proteins (K1578 and K1599) or increased potency for AChE (K1592 and K1594) were studied to identify the most promising pro-cognitive compound. Their effects were studied in cholinergic (scopolamine-induced) and glutamatergic (MK-801-induced) rat models of cognitive deficits in the Morris water maze. Moreover, the impacts on locomotion in the open field and AChE activity in relevant brain structures were investigated. The effect of the most promising compound on NMDA receptors was explored by in vitro electrophysiology. The cholinergic antagonist scopolamine induced a deficit in memory acquisition, however, it was unaffected by the compounds, and a deficit in reversal learning that was alleviated by K1578 and K1599. K1578 and K1599 significantly inhibited AChE in the striatum, potentially explaining the behavioral observations. The glutamatergic antagonist dizocilpine (MK-801) induced a deficit in memory acquisition, which was alleviated by K1599. K1599 also mitigated the MK-801-induced hyperlocomotion in the open field. In vitro patch-clamp corroborated the K1599-associated NMDA receptor inhibitory effect. K1599 emerged as the most promising compound, demonstrating pro-cognitive efficacy in both models, consistent with intended dual effect. We conclude that tacrine has the potential for development of derivatives with dual in vivo effects. Our findings contributed to the elucidation of the structural and functional properties of tacrine derivatives associated with optimal in vivo pro-cognitive efficacy.

3rd Faculty of Medicine Charles University Ruska 87 Prague 10 100 00 Czech Republic

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

Department of Animal Physiology and Ethology Faculty of Natural Sciences Comenius University Ilkovicova 6 Bratislava 4 842 15 Slovak Republic

Department of Physiology Faculty of Science Charles University Prague Albertov 6 Prague 2 12843 Czech Republic

Department of Toxicology and Military Pharmacy Military Faculty of Medicine University of Defence Trebesska 1575 Hradec Kralove 500 02 Czech Republic

Institute of Experimental Medicine of the Czech Academy of Sciences Videnska 1083 Prague 142 20 Czech Republic

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

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