A novel multistage antiplasmodial inhibitor targeting Plasmodium falciparum histone deacetylase 1

Although artemisinin combination therapies have succeeded in reducing the global burden of malaria, multidrug resistance of the deadliest malaria parasite, Plasmodium falciparum, is emerging worldwide. Innovative antimalarial drugs that kill all life-cycle stages of malaria parasites are urgently needed. Here, we report the discovery of the compound JX21108 with broad antiplasmodial activity against multiple life-cycle stages of malaria parasites. JX21108 was developed from chemical optimization of quisinostat, a histone deacetylase inhibitor. We identified P. falciparum histone deacetylase 1 (PfHDAC1), an epigenetic regulator essential for parasite growth and invasion, as a molecular target of JX21108. PfHDAC1 knockdown leads to the downregulation of essential parasite genes, which is highly consistent with the transcriptomic changes induced by JX21108 treatment. Collectively, our data support that PfHDAC1 is a potential drug target for overcoming multidrug resistance and that JX21108 treats malaria and blocks parasite transmission simultaneously.

The body weight of the tested mice from the experiment described in Fig. 2c. b In vivo activity of JX21108,(+)-JX21108 and (-)-JX21108 against the blood stage of P. yoelii in BALB/c mice. Five mice were used in each group. Parasitemia was calculated from at least 5,000 RBCs by microscopy. Blank, untreated group; PPQ, piperaquine phosphate. c and d The body weight and survival ratio of the tested mice from the experiment described in c. All data are the mean ± s.e.m.    was obtained by electrospray ionization (ESI) using a Waters GCT Premie and Waters LCT. HPLC analysis was performed on Agilent 1100 with quaternary pump and diode-array detector (DAD), using Agilent Exlipse XDB-C18 (250×4.6 mm, 5 µm particle size) column. Eluent was MeOH/aq. H3PO4 (0.1%, 90/10), flow rate was 0.5 ml/min, and peak purity was checked with UV spectra. Optical activity of enantiopure compound was measured via Rudolph Research Analytical AUTOPOL V automatic polarimeter.

Synthesis of linker derivatives a, b
Generally, preparation of linker derivatives is based on the synthetic procedure of JL01 1 using corresponding H-L-Boc as starting material. H-L-Boc were purchased from commercial suppliers.

General synthetic menthod of compounds JX21002-JX21022
General synthesis of S-1: To a mixture of corresponding H-L-Boc (1 g) and ethyl 2-chloropyrimidine-5-carboxylate (1.2 eq.) in DCM (0.2 M for H-L-Boc) was added DIPEA (1.5 eq.) in one portion at 0 °C under N2. The mixture was stirred at rt for 3 h. TLC (PE/EA = 2/1) showed the reaction was complete. The reaction mixture was concentrated under reduced pressure to remove DCM. The residue was purified by flash column chromatography (SiO2, PE/EA) to get compound S-1.

General synthesis of S-2:
To a solution of compound S-1 (1 eq) in DCM (0.5 M) was added HCl/1, 4-dioxane (4 M, 6 eq.). The mixture was stirred at rt for 2 h. TLC (PE/EA = 2/1) showed the reaction was complete. The solvent was removed in vacuo. The reaction mixture was washed with saturated NaHCO3 and filtered. The resulted precipitate was wash with H2O for 3 times, dried in vacuo to get deprotected mixture which was used directly in the next steps without purification.
To the mixture of deprotected mixture (considered as 1 eq.), N-methyl-3-formylindole (1.2 eq.) was added DCE (0.2 M) and HOAc (2 eq.) under N2. The mixture was stirred at rt for 30 min. NaBH(OAc)3 (1.5 eq.) was then added and the mixture stirred for 12 h. TLC (DCM/MeOH = 9/1) showed the reaction was complete. The reaction mixture was quenched by addition saturated aqueous NaHCO3, and then the reaction mixture was diluted with H2Oand extracted three times with DCM. The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (SiO2, DCM/MeOH) to get compound S-2.

General synthesis of S-3:
To a solution of compound S-2 (1 eq.) in THF (0.1 M) was added aqueous NaOH (1M, 10 eq.). The mixture was stirred at reflux until TLC (DCM/MeOH = 9/1) showed the reaction was complete. The mixture was then acidified to pH = 2~3 with 2 M HCl, causing a solid to precipitate. This solid was collected, washed with large amount of H2O to pH = ca.7 and dried to get hydrolyzed mixture which was used directly in the next steps without purification.
To the hydrolyzed mixture (considered as 1 eq.) was stirred with EDCI (3 eq.) and HOBt (3 eq.) in DMF (0.2 M) at rt under a nitrogen atmosphere for 30 min. THPONH2 (5 eq.) was then added followed by TEA (5 eq.) and the mixture allowed to stirred at rt for 72 h. TLC (DCM/MeOH = 9/1) showed the reaction was complete. The mixture was then diluted with H2O and extracted three times with DCM. The combined organic layers were wash twice with H2O, once with brine, dried (Na2SO4), and the solvent was removed in vacuo. The residue was purified by column chromatography (SiO2, DCM/MeOH) to get compound S-3.

General synthesis of compounds JX21002-JX21022
To a solution of compound S-3 (1 eq.) in DCM (0.1 M) was added HCl/dioxane (4 M, 2.5 eq.). The mixture was stirred at rt for 30 min under a nitrogen atmosphere. TLC showed the reaction was complete.
The reaction mixture was filtered and the resulted precipitate was wash with large amount of DCM and ether, dried in vacuo to get compound.

hydrochloride (JX21003)
Compound JX21003 was synthesized according to the above general synthetic method.

carboxamide hydrochloride (JX21010)
Compound JX21010 was synthesized according to the above general synthetic method.

carboxamide hydrochloride (JX21017)
Compound JX21017 was synthesized according to the above general synthetic method.

5-carboxamide hydrochloride (JX21022)
Compound JX21022 was synthesized according to the above general synthetic method.  General synthetic method of compounds JX21101-JX21143

Synthesis of S-4
To a solution of compound S-1-011(9.5 g, 25.4 mmol) in DCM (51 mL, 0.5 M) was added HCl/1, 4dioxane (4 M, 64 mL, 256 mmol, 10 eq.). The mixture was stirred at rt for 0.5 h. TLC (PE/EA = 2/1) showed the reaction was complete. The solvent was removed in vacuo. To the resultant mixture was added DCM (130 mL, 0.2 M), DIPEA (12.6 mL, 76.2 mmol, 3 eq.), then Cbz-Cl (4.4 mL, 30.8 mmol, 1.2 eq.) was added slowly to the mixture. The mixture was stirred at rt for 6 h. TLC (PE/EA = 2/1) showed the reaction was complete. The reaction mixture was poured into saturated NaHCO3 and the aqueous phase was extracted three times with DCM (300 mL in total). The combined organic layers was washed once with brine, dried (Na2SO4), and the solvent was removed in vacuo. The residue was purified by flash column chromatography (SiO2, PE/EA) to get compound S-4 (10 g, 96% yield

Synthesis of S-5
To a mixture of S-4 (10 g, 24.5 mmol), and potassium carbonate (8.5 g, 61.5 mmol, 2.5 eq.) was added H2O (60 mL) and MeOH (60 mL). The mixture was heated to 65 o C for 18 h. TLC (PE/EA = 2/1) showed the reaction was complete. The reaction mixture was concentrated under reduced pressure to remove solvent. The mixture was then acidified to pH = 2~3 with 2 M HCl, causing a solid to precipitate. This solid was collected, washed with large amount of H2O to pH = ca.7 and dried in vacuo. To the dried mixture was added HOBt (9.9 g, 73.5 mmol, 3 eq.), DCM (120 mL, 0.2 M) and stirred at rt under a nitrogen atmosphere until all the solid dissolved. EDCI (14 g, 73.5 mmol, 3 eq.) was added and stirred for 30 min. THPONH2 (5.7 g, 49 mmol, 2 eq.) and TEA (6.8 mL, 49 mmol, 2 eq.) and the mixture was allowed to stirred at rt for 72 h. TLC (DCM/MeOH = 9/1) showed the reaction was complete. The mixture was then diluted with H2O and extracted three times with DCM. The combined organic layers were wash twice with H2O, once with brine, dried (Na2SO4), and the solvent was removed in vacuo. The residue was purified by column chromatography (SiO2, DCM/MeOH) to get compound S-5 (10.6 g, 90% yield Synthesis of S-6 S-5 (0.48 g 1 mmol) was dissolved into DCM (5 mL) and MeOH (5 mL). To the solvent was added 10% Pd/C (0.19 g, wetted with ca. 55% water) and Et3SiH (96 μL, 0.6 mmol). The mixture was allowed to stirred at rt for 1 h, and Et3SiH (96 μL, 0.6 mmol) was added to the mixture. After 1 h, the mixture was filter through celite, and the solid residure was washed with MeOH. The solvent was rotavapored to dryness, and the residue was purified by column chromatography (SiO2, DCM/MeOH) to get compound S-6 (0.29 g, 84% yield). 1 H NMR ( and stirred for 24 h. The reaction mixture was poured into saturated NaHCO3 and the aqueous phase was extracted three times with DCM (75 mL in total). The combined organic layers was washed once with brine, dried (Na2SO4), and the solvent was removed in vacuo. The residue was purified by flash column chromatography (SiO2, DCM/MeOH) and the product was dissolved into DCM (0.05 M) under a nitrogen atmosphere and HCl/1, 4-dioxane (4 M, 2.4 eq.) was add in one portion. The mixture was stirred at rt for 30 min and TLC showed the reaction was complete. The reaction mixture was filtered and the resulted precipitate was wash with large amount of DCM and ether, dried in vacuo to get compound.

hydrochloride (JX21110)
Compound JX21110 was synthesized according to the above general synthetic method.

hydrochloride (JX21114)
Compound JX21114 was synthesized according to the above general synthetic method.