Synthesis, Antiplasmodial, and Antileukemia Activity of Dihydroartemisinin-HDAC Inhibitor Hybrids as Multitarget Drugs

Abstract

Artemisinin-based combination therapies (ACTs) are the gold standard for the treatment of malaria, but the efficacy is threatened by the development of parasite resistance. Histone deacetylase inhibitors (HDACis) are an emerging new class of potential antiplasmodial drugs. In this work, we present the design, synthesis, and biological evaluation of a mini library of dihydroartemisinin-HDACi hybrid molecules. The screening of the hybrid molecules for their activity against selected human HDAC isoforms, asexual blood stage P. falciparum parasites, and a panel of leukemia cell lines delivered important structure-activity relationships. All synthesized compounds demonstrated potent activity against the 3D7 and Dd2 line of P. falciparum with IC₅₀ values in the single-digit nanomolar range. Furthermore, the hybrid (α)-7c displayed improved activity against artemisinin-resistant parasites compared to dihydroartemisinin. The screening of the compounds against five cell lines from different leukemia entities revealed that all hydroxamate-based hybrids (7a-e) and the ortho-aminoanilide 8 exceeded the antiproliferative activity of dihydroartemisinin in four out of five cell lines. Taken together, this series of hybrid molecules represents an excellent starting point toward the development of antimalarial and antileukemia drug leads.

Keywords: artemisinin; histone deacetylase; multitarget drugs.

Figure 1

(A) HDACi pharmacophore model illustrated on the FDA-approved drug vorinostat (SAHA). (B) Chemical structure of dihydroartemisinin (DHA). (C) Compound design based on DHA as cap connected by C-10 ether or thioether groups to four different linkers and three zinc-binding groups.

Scheme 1

Synthesis of DHA–HDACi hybrids: (a) NaBH₄ (2.5 equiv.), MeOH, 0 °C → r.t., 0.5 h, 98%. (b) Ac₂O, pyridine, r.t., 20 h, 92% (c) HX-linker-COOH (1.2 equiv.), BF₃ OEt₂ (1.05 equiv.), DCM, −15 °C, 0.5 h, 55–75%. (d) NH₂-O-THP (1.0 equiv.), EDC.HCl (1.0 equiv.), DMAP (0.5 equiv.), DCM, r.t., 6 h. (e) benzoylchloride (cat.), EtOH, 0 °C, 3 h, 17–35% (over 2 steps). (f) o-phenylenediamine (1.0 equiv.), EDC.HCl (1.05 equiv.), DMAP (0.5 equiv.), DCM, r.t., 6 h, 24%.

Figure 2

K562 cells were treated with DHA, (β)-7c, and vorinostat (0.4 µM) for 24 h. Subsequently, cell lysates were immunobloted with anti-acetyl-α-tubulin and acetyl-histone H3 antibodies, whereas GAPDH served as a loading control. The experiments were repeated three times (n = 3), and a representative blot is shown here.

Figure 3

Docking pose of (α)-7c (A) and (β)-7c (B) in the catalytic domain 2 of HDAC6 (PDB: 5EDU [29]). Ligands are colored green and are depicted as sticks. The catalytic Zn²⁺-ion is shown as a gray sphere, and water is shown as a red sphere. The protein backbone is shown as light blue cartoon including the wheat-colored protein surface surrounding the ligand. The binding interactions of the hydroxamic acids are depicted as yellow, dashed lines.

Figure 4

Survival of the artemisinin-resistant (Dd2 R539T) and sensitive (Dd2) P. falciparum line after treatment with 700 nM of the respective compound (DHA or DHA–HDACi hybrid) in the ring-stage survival assay. Results show the survival in percent of the drug-treated parasites in relation to the untreated control (DMSO) parasites. Each experiment was performed twice in duplicate. All compounds, except 7a, were slightly more active, with (α)-7c being significantly more active than DHA alone (unpaired t-test: ** p < 0.005).

Figure 5

Apoptosis assay via annexin V/PI measurement after 48 h treatment of K562 cells with the respective inhibitors at the depicted concentrations. Significance was calculated using the sum of (early and late) apoptotic cells and necrotic cells vs. vehicle control (DMSO) from three independent measurements, using one-way ANOVA test (nonsignificant or ns, *** p < 0.0005 and **** p < 0.0001).