Synthesis of imidazopyridazine and pyrazolopyrimidine derivatives as potential inhibitors of Plasmodium kinases P14K and PKG

Abstract

Malaria is a disease that is caused by various Plasmodium species, with Plasmodium falciparum and vivax being the most prevalent. The disease is usually mostly severe in pregnant women and children under the age of five. The 2020 World Malaria Report from WHO estimated 241 million new malaria cases and 627000 malaria deaths globally. In comparison to 2019, there has been an increase of approximately 14 million new cases and 69000 deaths. Approximately 47000 of these additional deaths were caused by interruptions in malaria diagnosis, prevention, and treatment during the COVID-19 pandemic. Emergence of resistance towards previously effective anti-malarial drugs, has resulted in an urgent need for the development of new drugs with new modes of action. A series of imidazopyridazine and pyrazolopyrimidine derivatives have been reported to be potent against sensitive (NF54) strain of the human malaria parasite Plasmodium falciparum. In this study we focused on synthesizing a new library of imidazopyridazine and pyrazolopyrimidine derivatives, with substituents at 3- & 6- positions and 3- & 5- positions respectively. The compounds were successfully synthesized and characterized by NMR and HRMS. The imidazopyridazine derivatives percentage yields ranged from 38% to 70%, whereas the percentage yields for the pyrazolopyrimidine derivatives ranged from 41% to 75%. All the imidazopyridazine compounds were evaluated for their in vitro antiplasmodial activities against NF54 strain and inhibitory activity against Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) and Plasmodium vivax phosphatidylinositoil 4-kinase type III beta (PvPI4Kβ). The pyrazolopyrimidine compounds were only evaluated for their in vitro antiplasmodial activity against the NF54 strain. Imidazopyridazine compounds 20B, 20D, 20E, 20F, 20M and 20O demonstrated in vitro antiplasmodial activity of below 6 𝜇M against NF54 strain, which was used as a cutoff to determine good activity versus moderate activity. Imidazopyridazine compounds 20A, 20C, 20G, 20H, 20I, 20J, 20K, 20L and 20N demonstrated a moderate activity of greater than 6 𝜇M against NF54 strain. Imidazopyridazine compounds 20H, 20K, 20L and 20N demonstrated good activity against both PfPKG and PvPI4Kβ with IC50 values ranging from 0.96 𝜇M to 5.04 𝜇M and 0.006 𝜇M to 0.071 𝜇M, respectively. Imidazopyridazine compounds 20A, 20B, 20C, 20D, 20E, 20F, 20G, 20I, 20J, 20M and 20O demonstrated moderate activity of greater than 10 𝜇M against PfPKG and good activity against PvPI4Kβ with IC50 values ranging from 0.006 𝜇M to 0.643 𝜇M. From the pyrazolopyrimidine scaffold, compounds 39A (0.013 𝜇M) and 39E (0.009 𝜇M) demonstrated the highest activities against the NF54 strain. The reduction of the pyrazolopyrimidine scaffold did not improve the potency, as it was seen with compounds 39A (0.013 𝜇M) reduced to 40A (0.197 𝜇M), compound 39B (0.190 𝜇M) reduced to 40B (1.056 𝜇M) and compound 39C (1.504 𝜇M) reduced to 40C (2.113 𝜇M). 3-(Methylsulfonyl) phenyl was a better substituent to have on the left-hand side of the pyrazolopyrimidine core scaffold than 4-(methylsulfonyl) phenyl substituent. Pyrazolopyrimidine compounds demonstrated very good in vitro antiplasmodial activity against the sensitive (NF54) strain as compared to the imidazopyridazine compounds. Therefore, pyrazolopyrimidine is a better scaffold to explore to eradicate malaria. Compounds 38A, 38A, 39B, 39D and 39E have a selectivity index of more than 100, therefore they will be progressed further for solubility, chemical stability, microsomal stability (m,r,h) and in vitro PfK1, Dd2 assays