Development of Potent Pf CLK3 Inhibitors Based on TCMDC-135051 as a New Class of Antimalarials

Abstract

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.

Figure 1

Structure and biological profile of hit PfCLK3 inhibitor TCMDC-135051, 1.

Figure 2

(A) Aurora kinase inhibitor GSK1070916, (B) 1-p-chlorobenzyl-7-azaindole-3-α-piperidylmethanol has in vivo activity against P. berghei, (C) PfHsp90 inhibitor IND31119, and (D) drug candidates incorporating a core 7-azaindole scaffold, vemurafenib, PLX3397, and AZD5363.

Scheme 1. Synthesis of TCMDC-135051 1 and Analogues 8a–c & 9

Reagents and conditions: (i) TsCl, NaH, THF, 0 °C, 2 h; (ii) LDA, I₂, THF, −78 °C, 3 h; (iii) (5-formyl-2-methoxyphenyl)boronic acid, Pd(PPh₃)₄, Na₂CO₃, 1,4-dioxane, 110 °C, 12 h; (iv) amine, NaBH(AcO)₃, 1,4-dioxane, 20 °C, 12 h; (v) CH₃OH, K₂CO₃, 55 °C, 18 h; (vi) 2-isopropyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid, Pd(dppf)Cl₂·CH₂Cl₂, Na₂CO₃, 1,4-dioxane, 110 °C, 0.5 h, MW; and (vii) SOCl₂, CH₃CH₂OH, reflux, 18 h.

Scheme 2. Synthesis of 4-(2-(5-(Aminomethyl)-2-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)-2-isopropylbenzoic Acid 12

Reagents and conditions: (i) 4-methoxy-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzonitrile, Pd(PPh₃)₄, Na₂CO₃, 1,4-dioxane, 110 °C, 18 h; (ii) CH₃OH, K₂CO₃, 55 °C, 18 h; (iii) 2-isopropyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid, Pd(dppf)Cl₂.CH₂Cl₂, Na₂CO₃, 1,4-dioxane, 110 °C, 0.5 h, MW; and (iv) CoCl₂·6H₂O, NaBH₄, CH₃OH.

Scheme 3. Synthesis of 2-Isopropyl-4-(2-(2-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)benzoic Acid 15, 4-(2-(5-((Diethylamino)methyl)-2-hydroxyphenyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)-2-isopropylbenzoic Acid 19, 4-(2-(3-((Diethylamino)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)-2-isopropylbenzoic Acid 23 and [4-(2-(3-((Diethylamino)methyl)-2-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)-2-isopropylbenzoic Acid] 27

Reagents and conditions: (i) (2-methoxyphenyl)boronic acid, Pd(PPh₃)₄, Na₂CO₃, 1,4-dioxane, 110 °C, 12 h; (ii) CH₃OH, K₂CO₃, 55 °C, 18 h; (iii) 2-isopropyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid, Pd(dppf)Cl₂·CH₂Cl₂, Na₂CO₃, 1,4-dioxane, 110 °C, 0.5 h, MW; (iv) 4-(methoxymethoxy)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzaldehyde or 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzaldehyde, Pd(PPh₃)₄, Na₂CO₃, 1,4-dioxane, 110 °C, 18 h; (v) Et₂NH, NaBH(AcO)₃, 1,4-dioxane, rt, 18 h; and (vi) HCl, MeCN/H₂O 3:1.

Scheme 4. Synthesis of 4-Aryl-7-azaindole Analogues, 28–32

Reagents and conditions: (i) boronate esters, Pd(dppf)Cl₂.CH₂Cl₂, Na₂CO₃, 1-4-dioxane, 110 °C, 0.5 h, MW.

Figure 3

Putative binding mode of TCMDC-135051 1 in a PfCLK3 homology model.

Figure 4

PRR of clinical isolates comparing pyrimethamine and TCMDC-135051 at EC₅₀ and 10 times the EC₅₀ of each drug. This represent the average of triplicate.