Development of Potent PfCLK3 Inhibitors Based on TCMDC-135051 as a New Class of Antimalarials

The protein kinase PfCLK3 plays a critical role in the regulation of malarial parasite RNA splicing and is essential for the survival of blood stage Plasmodium falciparum. We recently validated PfCLK3 as a drug target in malaria that offers prophylactic, transmission blocking, and curative potential. Herein, we describe the synthesis of our initial hit TCMDC-135051 (1) and efforts to establish a structure–activity relationship with a 7-azaindole-based series. A total of 14 analogues were assessed in a time-resolved fluorescence energy transfer assay against the full-length recombinant protein kinase PfCLK3, and 11 analogues were further assessed in asexual 3D7 (chloroquine-sensitive) strains of P. falciparum parasites. SAR relating to rings A and B was established. These data together with analysis of activity against parasites collected from patients in the field suggest that TCMDC-135051 (1) is a promising lead compound for the development of new antimalarials with a novel mechanism of action targeting PfCLK3.


I. General information
Chemicals and solvents were purchased from standard suppliers and used without additional purification. All glassware was dried with a flame under flushing argon gas S6 or stored in the oven and let cool under an inert atmosphere prior to use. Anhydrous solvents (THF, DCM and Et2O) were obtained by passage through solvent filtration systems (Pure Solv) and solvents were transferred by syringe. PET ether refers to petroleum (bp. 40-60 °C, reagent grade, Fisher Scientific). All reactions carried out under inert or dry atmosphere were carried out under a blanket of nitrogen. Thinlayer chromatography (TLC) was performed using aluminium plates precoated with silica gel (0.25 mm, 60 A° pore-size) impregnated with a fluorescent indicator (254 nm).Visualization on TLC was achieved by the use of UV light (254 nm). Flash column chromatography was undertaken on silica gel (400-630 mesh). Proton

Method B: Reductive Amination of Aldehydes
To a solution of aryl aldehyde (1.0 equiv) in 1,4-dioxane was added amine (1.5 equiv) and the solution was allowed to stir for 2 mins before the addition of sodium triacetoxyborohydride (2.5 equiv). The reaction mixture was stirred at room temperature for 18 h before quenching with ammonium hydroxide. The reaction mixture was extracted with ethyl acetate and washed with brine. The organic layer was dried over magnesium sulphate and the residue purified by flash column chromatography as indicated.

Method C: Deprotection of azaindole
To a solution of protected 7-azaindole (1 equiv) in methanol was added potassium carbonate (3.5 equiv) and refluxed for 18 h. Poured the reaction into a mixture of EtOAc (10 mL) and H2O in a separatory funnel. Solvent was then removed under vacuum and the residue was then purified by flash column chromatography as indicated.

Method D: Suzuki Cross-Coupling with boronate ester
To a 10 mL microwave vial containing the required bromo-7-azaindole (1 equiv) in 1,4-dioxane was added boronic acid/ester (1.1 equiv), Pd(dppf)Cl2·DCM complex (0.05 equiv.) under a nitrogen atmosphere. The solution was purged with nitrogen for 5 mins and the reaction microwaved at 110 ºC for 0.5 h. The reaction was allowed to cool to room temperature and the mixture was filtered through celite eluting with methanol. The filtrate was evaporated and the resulting residue was purified by preparative HPLC: 10-95% acetonitrile in water + 0.1% TFA to give the desired products.

Method E: Synthesis of boronate ester
Boronate esters required for Suzuki coupling were prepared according to procedure reported in literature. 1 To a solution of aryl bromide (1 equiv), bis(pinacolato)diboron (1.5 equiv) and potassium acetate (3 equiv) in 1,4-dioxane (20 ml), PdCI2(dppf)-CH2Cl2 complex (0.1 equiv) were added under nitrogen and stirred at 100 °C for 3 hour. The reaction mixture was quenched with saturated NaHCO3 and extracted with ethyl acetate. The organic phase was dried (Na2SO4), filtered and concentrated to dryness. The crude product was purified by chromatography (20% ethyl acetate-PET Ether) to give the desired boronate esters.

P. falciparum culture and synchronisation
P. falciparum cultures were maintained in RPMI-1640 media (Invitrogen) supplemented with 0.2% sodium bicarbonate, 0.5% Albumax II, 2.0 mM L-glutamine (Sigma) and 10 mg/L gentamycin. For continuous culture, the parasites were kept at 4% haematocrit in human erythrocytes from 0+ blood donors and between 0.5 -3% parasitaemia maintained in an incubator at 37 °C , 5% carbon dioxide (CO2), 5% oxygen (O2) and 90% nitrogen (N2). To obtain highly synchronous ring stage parasites for the drug assays, cultures were double synchronised using Percoll and Sorbitol synchronisation as previously described. [2][3] First, highly segmented schizonts were enriched by centrifugation on a 70% Percoll (GE Healthcare) cushion gradient. The Schizont pellet was collected and washed before fresh erythrocytes were added to a final haematocrit of 4%. The schizonts were incubated for about 1-2 hours shaking continuously to allow egress and re-invasion of new erythrocytes.
Residual schizonts were then removed by treating the pellet with sorbitol to generate highly synchronous 1-2 hours old ring-stage parasites.

Determining the IC 50 of compound inhibitors and drugs-ex vivo
To determine the IC50 of the molecules in parasites (P. falciparum 3D7) ex vivo, the The data was normalised against the controls and graphs were generated using Graph Pad Prism 8 to determine the IC50 values using the non-linear regression log (inhibitor) versus response (three parameter) curve.

Time Resolve Florescence Energy Transfer (TR-FRET) to determine the IC 50 of the inhibitors with full-length PfCLK3 recombinant protein
The TR-FRET assays, a high-throughput inhibition assay, as described previously 4 was used to determine the potency of the small molecules generated against full-