Ring Distortion of Vincamine Leads to the Identification of Re-Engineered Antiplasmodial Agents

Abstract

There is a significant need for new agents to combat malaria, which resulted in ∼409,000 deaths globally in 2019. We utilized a ring distortion strategy to create complex and diverse compounds from vincamine with the goal of discovering molecules with re-engineered biological activities. We found compound 8 (V3b) to target chloroquine-resistant Plasmodium falciparum Dd2 parasites (EC₅₀ = 1.81 ± 0.09 μM against Dd2 parasites; EC₅₀ > 40 μM against HepG2 cells) and established structure-activity relationships for 25 related analogues. New analogue 30 (V3ss, Dd2, EC₅₀ = 0.25 ± 0.004 μM; HepG2, EC₅₀ > 25 μM) was found to demonstrate the most potent activity, which prevents exit on the parasite from the schizont stage of intraerythrocytic development and requires >24 h to kill P. falciparum Dd2 cells. These findings demonstrate the potential that vincamine ring distortion has toward the discovery of novel antimalarial agents and other therapies significant to human health.

Figure 1

Complex and diverse scaffolds rapidly synthesized from vincamine. In this study, analogues 8 (V3b) and 7 (V4g) were identified as hit compounds demonstrating antiplasmodial activities against Dd2 parasites (note: vincamine does not have antiplasmodial activity).

Figure 2

(A) Antiplasmodial activities of V3 analogues from initial investigations with chloroquine-resistant P. falciparum parasites. (B) Future analogues were informed by initial antiplasmodial findings coupled to structural modifications of 8 (V3b) that could be probed with iterative rounds of synthesis and in vitro testing.

Figure 3

(A) Scale-up efforts to key ring-cleaved building block 8 (V3b). (B) Chemical synthesis of V3 analogues for initial optimization and to establish an SAR profile. (C) Chemical synthesis of 7 (V4g).

Figure 4

Detailed SAR profiles regarding the V3 antiplasmodial agents.

Figure 5

Stage-specific activity of 30 (V3ss) in P. falciparum Dd2 cells. Tightly synchronized Dd2 cultures were treated with 5 x EC₅₀ of 30 at 6, 18, 30, and 42 HPI. Blood smears and Giemsa staining were taken at each time point as well to verify the phenotype. Flow cytometry was performed to quantify the stage of action for V3ss at the time of harvesting, following 6, 18, 30, and 42 HPI treatments. For flow cytometry analysis, collected samples were fixed in a freshly prepared solution of 4% paraformaldehyde and 0.0075% glutaraldehyde, permeabilized with 0.25% Triton X-100, treated with RNase (50 μg/mL), and stained with YOYO-1. Data acquisition was performed in a CytoFLEX (Beckman) flow cytometer. DHA and atovaquone were evaluated as positive controls in stage-specific activity assays, and results from these experiments can be viewed in Supporting Information. Parasites following exposure to 30 (V3ss) are arrested at the late schizont stage.

Figure 6

Kinetic kill experiments used to determine parasitocidal/parasitostatic activity of 30/V3ss in P. falciparum Dd2 cultures. Asynchronous cultures were treated with 30 at 5 × EC₅₀ for (A) 6, (B) 12, (C) 24, and (D) 48 h. Following each treatment, cultures were washed three times in RPMI, then resuspended in culture media, and monitored daily for parasite growth. Note: Dihydroartemisinin (DHA) was used as a positive control for rapid parasite killing (5 × EC₅₀ = 50 nM) and atovaquone was used as a positive control for slow parasite killing (5 × EC₅₀ = 6.6 nM) in these experiments. DMSO concentration was 0.15% in kinetic kill experiments.