Towards Novel Antiplasmodial Agents—Design, Synthesis and Antimalarial Activity of Second-Generation β-Carboline/Chloroquine Hybrids

As the resistance of <i>Plasmodium</i> to the existing antimalarials increases, there is a crucial need to expand the antimalarial drug pipeline. We recently identified potent antimalarial compounds, namely harmiquins, hybrids derived from the β-carboline alkaloid harmine and 4-amino-7-chloroquinoline, a key structural motif of chloroquine (CQ). To further explore the structure-activity relationship, we synthesised 13 novel hybrid compounds at the position <i>N</i>-9 of the β-carboline ring and evaluated their efficacy in vitro against <i>Plasmodium falciparum</i> 3D7 and Dd2 strains (CQ sensitive and multi-drug resistant, respectively). All compounds exhibit persistent antimalarial activity against both strains of <i>P. falciparum</i>. The most interesting derivatives had low nanomolar activity against both strains (IC₅₀ (<b>33</b>) = 4.7 ± 1.3 nM against <i>Pf</i>3D7 and 6.5 ± 2.5 nM against <i>Pf</i>Dd2; IC₅₀ (<b>37</b>) = 4.6 ± 0.6 nM against 3D7 and 10.5 ± 0.4 nM against Dd2). Resistance indices (RIs) ranged from 0.9 to 5.3 compared to CQ (RI = 14.4), highlighting their superior consistency in activity against both strains. The cytotoxicity screening performed on HepG2 revealed over 3 orders of magnitude higher IC₅₀ for most of the compounds, with SIs from 711.0 to 8081.8. Spectroscopic studies explored the affinities of newly synthesised compounds for DNA, RNA, and HSA. Both tested hybrids, <b>34</b> and <b>39</b>, were intrinsically fluorescent in an aqueous medium, characterised by remarkable Stokes shifts of emission maxima (Δ<i>λ</i> = +103 and +93 nm for <b>34</b> and <b>39</b>, respectively). Fluorimetric experiments revealed that compound <b>34</b>, with its shorter and more flexible linker, exhibited at least an order of magnitude higher affinity toward ds-DNAs versus ds-RNA and two orders of magnitude higher affinity toward GC-DNAs compared to <b>39</b>. The behaviour of the investigated compounds upon binding to HSA is very similar, showing a strong hypsochromic shift of the emission maximum (almost Δ<i>λ</i> = -70 nm) and demonstrating their effectiveness as fluorimetric probes for distinguishing between DNA/RNA and proteins.