Antiplasmodial Activity and In Vivo Bio-Distribution of Chloroquine Molecules Released with a 4-(4-Ethynylphenyl)-Triazole Moiety from Organometallo-Cobalamins

Abstract

We have explored the possibility of using organometallic derivatives of cobalamin as a scaffold for the delivery of the same antimalarial drug to both erythro- and hepatocytes. This hybrid molecule approach, intended as a possible tool for the development of multi-stage antimalarial agents, pivots on the preparation of azide-functionalized drugs which, after coupling to the vitamin, are released with a 4-(4-ethynylphenyl)-triazole functionality. Three chloroquine and one imidazolopiperazine derivative (based on the KAF156 structure) were selected as model drugs. One hybrid chloroquine conjugate was extensively studied via fluorescent labelling for in vitro and in vivo bio-distribution studies and gave proof-of-concept for the design. It showed no toxicity in vivo (zebrafish model) as well as no hepatotoxicity, no cardiotoxicity or developmental toxicity of the embryos. All 4-(4-ethynylphenyl)-triazole derivatives of chloroquine were equally active against chloroquine-resistant (CQR) and chloroquine-sensitive (CQS) Plasmodium falciparum strains.

Keywords: antimalarial; chloroquine; cobalamin; in vivo; prodrug; triazole; zebrafish model.

Figure 1

Molecular structures of antimalarial drugs mentioned in the introduction. JR1-3 and SN1 are the molecules designed for this study (vide infra for details). The red box indicates the structural feature that we took as reference for the design of the compounds.

Figure 2

General synthetic scheme for the synthesis of derivatives B₁₂-JR1-JR3 and B₁₂-SN1.

Figure 3

Left, aromatic region of the ¹H-NMR spectra (D₂O, 500 MHz) of CN-Cbl and of the derivatives B₁₂-JR1 to -JR3. Right, crystal structure of compound B₁₂-F2 and of JR1 (thermal ellipsoids are shown at the 50% probability).

Figure 4

(A) Reaction steps leading to the synthesis of the green emitting B₁₂-JR1-CBC. Conditions: (i) CDT, 12 h, DMSO; (ii) PEG, 12 h, anhydrous DMF; (iii) NHS-rhodamine, 12 h, TEA, anhydrous DMF. (B) Normalized excitation and emission profile of B₁₂-JR1-CBC in a 1:1 H₂O/MeOH solution. (C,D) Fluorescent microscope images of erythrocytes within full canine blood smear incubated with B₁₂-JR1-CBC at 37 °C (GFP filter; 100 × magnification). (E) Conceptual drawing of B₁₂-JR1-CBC accumulation within erythrocytes. Note: as a full blood smear was made, fluorescence around red blood cells is due to serum and/or burst erythrocytes.

Figure 5

(A) Spectroscopic change observed in the Soret band of PPIX when it is titrated with drugs presented in this study. (B) Variation in absorbance of PPIX at 402 nm as a function of drug concentration. (C) Visible spectra of PPIX in 40% aqueous DMSO, pH 7.5 before (black line) and after addition of excess drug (red line). The spectrum of the latter is identical to that of the μ-oxo dimer previously reported. Note the peak maximum at 576 nm. (D) Density Functional Theory (DFT) optimized structure (gas-phase) of the interaction of a protonated 4-(4-ethynylphenyl)-triazole functionalized quinoline drug model with ferriprotoporphyrin IX μ-oxo dimer.

Figure 6

Toxicity evaluation of B₁₂-JR1 and B₁₂-JR1-CBC in the zebrafish model over a period from 6 to 120 hpf, expressed as the LC₅₀ values. Shown are data relative to (A) embryos survival/teratogenicity and (B) cardiotoxicity. The liver is outlined with a dashed line.

Figure 7

Bio-distribution and toxicity evaluation of B₁₂-JR1 and B₁₂-JR1-CBC in the 120-hpf old transgenic Tg(fabp10:EGFP) zebrafish embryo with fluorescently labelled liver. Panel 1 (top left). Top: accumulation of B₁₂-JR1 (150 µM) in the liver applied at 106 hpf in the embryo water. (A) Embryo imaged by a fluorescent microscope with a filter enabling only EGFP-labelled liver visualization. (B) Embryo imaged with a filter enabling B₁₂-JR1 visualization, but not EGFP-labelled liver. (C) Merged images A and B. Middle: accumulation of B₁₂-JR1-CBC (20 µM) in the liver applied at 106 hpf in the embryo water. (D) Embryo imaged upon a filter enabling only EGFP-labelled liver visualization. (E) Embryo imaged with a filter enabling only B₁₂-JR1 visualization, and (F) merged Tg(fabp10:EGFP) and B₁₂-JR1-CBC fluorescent signals in the same embryo. Bottom: an accumulation of B₁₂-JR1-CBC (47.8 µg per an embryo, corresponding to 20 µM dose) in the liver microinjected (parenteral use) into embryo’s circulation at 106 hpf. Arrows in the panel 1 indicate the liver position within embryos body. Panel 2 (top right): distribution of B₁₂-JR1 (150 µM) within the 120-hpf old embryos when applied at 6 hpf (the liver-free stage, A) or 72 hpf (the stage with functional liver, B). Lateral views (C and D) of the 120-hpf embryo with an overlay. Overlay outlines the pharynx (Ph), esophagus (E), liver (L), gallbladder (G), pancreas (P), swimmbladder (SB), and intestine (I). * marks intestinal lumen. Panel 3 (bottom). Hepatotoxicity evaluation in the transgenic Tg(fabp10:EGFP) zebrafish embryos with EGFP-labelled liver after the embryos exposure to the tested compounds in a period from 72–120 hpf (A). The liver area index was assessed in 120-h old zebrafish embryos (B) and indicated no changes in the treated groups compared to the DMSO-treated (control) one (n = 15).