Hit to Lead Stage Optimization of Orally Efficacious β-Carboline Antimalarials

Malaria, a disease caused by the parasite Plasmodium, continues to be one of the deadliest diseases worldwide. The WHO reported over 627,000 deaths in 2020, and over 1 billion people are at risk of infection. Even though Artemisinin-based Combination Therapies (ACT) are the current standard of care for malaria, the emergence of drug resistance generates a constant need to develop and synthesize new drugs. Tetrahydro-β-carboline acid (THβC) 1-(2,4-dichlorophenyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-2-ium-3-carboxylate (MMV008138) has promising antimalarial properties; it was discovered by screening the Malaria Box with the so-called IPP Rescue assay. This assay identified MMV008138 as an inhibitor of the MEP pathway, which produces essential isoprenoid precursors (IPP and DMAPP) in the malaria parasite P. falciparum (EC50 250 ± 70 nM, IPP rescue 100% @ 2.5 μM). Subsequent investigation revealed that (1R,3S)-configuration and 2',4'-dihalogen substitution were critical for the activity of this compound, and that substitution of the non-aromatic ring was not tolerated. To search for new antimalarial structures, our collaborator Dr. Max Totrov constructed a generalized 3D pharmacophore-based on MMV008138 and 92 of its analogs and used it for a virtual ligand screen (VLS) of the 13K compound hit set from which MMV008138 had been selected. This exercise identified TCMDC-140230, a THβC, 1-(3,4-dichlorophenyl)-8-methyl-N-(2-(methylamino)ethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxamide (undefined stereochemistry) reported having nearly the same potency of MMV008138. Synthesis of the stereoisomers of compound TCMDC-140230 was accomplished via Pictet-Spengler reaction of (S)- and (R)-7-methyl tryptophan methyl ester and 3,4-dichlorobenzaldehyde. The individual stereoisomeric esters were converted to the corresponding amides, but none of the stereoisomers of TCMDC-140230 were potent antimalarials (IC50 = 1,300 – 3,700 nM). However, a significant amount of oxidized byproduct 1-(3,4-dichlorophenyl)-8-methyl-N-(2-(methylamino)ethyl)-9H-pyrido[3,4-b]indole-3-carboxamide (MMV1803522) was observed in the synthesis of (1S,3S)- and (1R,3R)-TCDMC-140230. This achiral β-carboline amide (PRC1584, IC50 = 108 ± 7 nM) proved more potent towards P. falciparum than MMV008138 and its toxicity was not reversed by co-application of IPP. Thus, the antimalarial target of MMV1803522 is distinct from that of MMV008138. Most importantly, MMV1803522 at 40 mg/kg/day (oral) cured P. berghei malaria infection in mice. The lead compound also was found to have a good safety profile. Medicines for Malaria Venture (MMV) has expressed interest in this compound which is now also known as MMV1803522.
The results from these biological assays gave the insight to develop new analogs that have better asexual blood stage inhibition potency. Extensive structure-activity relationship studies were conducted by synthesizing analogs of the compound MMV1803522. The studies were mainly focused on analyzing the effect of aliphatic substitutions, how well the potency can be improved with different D-ring substitutions, and amide substitutions. In addition to this structural optimization, several metabolism studies were also conducted on this new lead compound. The potency study results of C1 alkyl-substituted analogs of MMV1803522 showed that aromatic substitutions are required at C1 for maintaining good inhibition potency. The heteroaryl substituents at C1 were found to be slightly less potent than the lead compound MMV1803522. Synthesis of analogs without C8 methyl group as in lead compound showed an EC50 < 100 nM is possible with a C8 hydrogen substitution. Most noteworthy is 3,4,5-trichlotophenyl-bearing compound 3.20a, which had an EC50 of 54 ± 8 nM. This compound is twice as potent as MMV1803522. Equipotent analogs to MMV1803522 were also synthesized with different amide substituents. The metabolism studies showed low solubility for compounds having an EC50 less than or close to 100 nM. Unfortunately, the intrinsic clearance rate of several selected compounds was found to be higher than MMV1803522. These results left us with scope for the development of new analog compounds. The emerging structure-activity relationship within this scaffold and outline of remaining challenges to improve potency sub-100 nM without compromising moderate solubility and good metabolic stability are in progress.