Translational (human volunteers)

Upon the completion of preclinical trials, the drug will have either passed or failed the required safety standards and pharmacokinetic profiling. For a compound that has passed these requirements, trials may then be conducted in human volunteers in order to show its efficacy as a potential treatment.

P218 (Fig. 18), discovered by BIOTEC Thailand in 2012, is an antifolate antimalarial drug bearing resemblance to the 2,4-diaminopyrimidine core structure of PYR, and is highly selective for the P. falciparum dihydrofolate reductase (Pf DHFR) [98].

Fig. 18

Key biological and physical properties of P218.

Unlike the typical high-throughput screening that is used for the identification of hit compounds in medicinal chemistry, P218 was identified through careful examination of the cocrystal structures of known Pf DHFR inhibitors and their substrates. The initial observation that 2,4-diaminopyrimidines acted as antagonists to folic acid led to the discovery and development of methotrexate (MTX) as an antitumor drug (Fig. 19). By examining the binding interactions with DHFR, the structure of P65 was identified. A total of over 200 compounds (not described in the original report) were designed and synthesized after further examination of potential interactions with amino acid residues, with the optimized structure of P218 being identified as the best compound.

Fig. 19

Key compounds in the discovery of P218. The key 2,4-diaminopyrimidine core highlighted in red can be found in a number of DHFR inhibitors.

The 2,4-diaminopyrimidine scaffold of P218 has been found to bind deep in the active site of Pf DHFR in both wild-type and mutant strains. This, along with the hydrogen bonding interaction of the carboxylate group with an Arg residue at the opposite end of the active site results in tighter binding and a longer residence time when compared to PYR. Since P218 is contained almost entirely within the dihydrofolate binding site, the strength of the binding should be strong enough to overcome any amino acid mutations, thus minimising the chance of drug-resistant mutations to arise. The novel two-step mechanism of action for binding to Pf DHFR (vide infra) allows P218 to overcome the resistance that has emerged from the use of pyrimethamine. P218 has also shown high selectivity to the binding of malarial over human DHFR, which translates into reduced toxicity.

In vivo studies have shown P218 to be highly efficacious against P. falciparum and Plasmodium chabaudi in mice with ED₉₀ values of 1 mg/kg and 0.75 mg/kg respectively. In the in vitro and in vivo potency assays that were run, P218 was found to be more potent than PYR in all cases.

Along with its high potency and good safety profile, P218 has the potential to be a replacement for PYR combination with CG in areas where Pf DHFR resistance has emerged. P218 has currently completed Phase I trials (NCT02885506).

Discovered by a partnership between St Jude Children’s Research Hospital and Rutgers University in 2010, (+)-SJ733 (Fig. 20) is a novel tetrahydroisoquinolone carboxanilide that possesses excellent anti-malarial activity in vivo [99].

Fig. 20

Key biological and physical properties of (+)-SJ733. Sexual stage potency and logD kindly provided by K. Guy, personal communication, 2018.

High-throughput phenotypic screening of > 300,000 compounds against the 3D7 strain of P. falciparum discovered a number of bioactive scaffold types, in which the hit compound (S′1, Fig. 21), belonging to the tetrahydroisoquinolone carboxanilide class, was found to have potent in vitro activity (EC₅₀ = 53 nM). Metabolic studies using mouse liver microsomes identified the susceptibility of the methoxy group to demethylation. In order to overcome this issue, the aniline substituents were replaced with fluoro and cyano groups (S′2), which had an added effect of improving the potency more than twofold. The thiophene group was suspected to be a metabolic hot spot, which was addressed by its replacement with a pyridyl group (S′3) resulting also in improved solubility while maintaining potency. The final metabolically labile group (isobutyl) was replaced with a trifluoromethyl moiety, further improving solubility and maintaining potency to give the lead compound (+)-SJ733. The (+)-(3S,4S) isomer was found to be significantly more potent (EC₅₀ = 36 nM) than its (–)-(3R,4R) enantiomer (EC₅₀ = 587 nM) [100].

Fig. 21

Key stages in the hit to lead pathway of (+)-SJ733. Poor metabolic stability of the hit compound was addressed by replacement of the chloro and methoxy groups with cyano and fluoro groups respectively. Further in vivo stability and solubility improvements were made by changing the thiophene to a pyridine. Finally, the gem-dimethyl group was substituted with a trifluoromethyl group to eliminate possible metabolic oxidation.

In the P. berghei mouse model, (+)-SJ733 was found able to cure malaria at doses of 4 × 100 mg/kg and has an ED₉₀ of 1.9 mg/kg. It has shown transmission-blocking activity in infected mice with an ED₅₀ of 5 mg/kg. It possesses a good safety profile with no cytotoxicity and was found to be more potent in vivo when compared to existing antimalarials such as artesunate, chloroquine, and pyrimethamine [101].

The molecular target of (+)-SJ733 has been identified as the P-type Na⁺–ATPase transporter (Pf ATP4, vide infra), which has been implicated as a target for a number of other structurally diverse compounds [102]. The compound is currently in the recruiting stage of first-in-human study trials (NCT02661373).

ACT-451840 (Fig. 22) is a phenylalanine-based compound that was developed in 2016 through a collaboration between Actelion Pharmaceuticals and the Swiss Tropical and Public Health Institute (STPHI). It has the potential to be a fast-acting drug with a long half-life and has shown efficacy against multiple stages of the P. falciparum parasite [103].

Fig. 22

Key biological and physical properties of ACT-451840.

Initial erythrocyte-based phenotypic screening of ∼ 5000 compounds identified A′1 (Fig. 23) as a highly potent compound against the chloroquine-resistant K1 strain of P. falciparum (IC₅₀ = 3.8 nM). The SAR studies in this project were unique in that the anti-malarial activity was measured in parallel in two different media: 10% bovine serum albumin and 50% human serum, with the latter, used to help identify any potential problems with protein binding at an early stage of the optimization. The stereogenic centre of the amino acid residue proved to be important for the activity, with the (S)-isomer showing more than tenfold higher activity compared to the non-natural (R)-isomer. Modification of the n-pentyl chain to an acylpiperazine group (A′2) resulted in an improvement in the physical and chemical properties of the compound. Replacement of the trifluoromethyl group with a tert-butyl group (A′3) led to improved antimalarial activity, especially in the presence of human serum proteins. Final installation of a 4-cyano moiety on the southern phenyl ring gave the highly potent (IC₅₀ = 0.4 nM) lead candidate ACT-451840.

Fig. 23

Key stages in the hit to lead pathway of ACT-451840. Initial change of the n-pentyl group to an acylpiperazine (A′2) helped to improve the physicochemical properties. The subsequent introduction of a tert-butyl in place of the trifluoromethyl (A′3) and a cyano group on the southern phenyl ring resulted in the optimized compound.

Interestingly, all compounds were found to be significantly less active against P. berghei than the human parasite. This is notable since most new anti-malarial drugs in development have shown similar potency against both rodent and human parasites. As a result, for in vivo studies, it was crucial to use a humanized P. falciparum severe combined immunodeficiency (SCID) mouse model. In the P. berghei mouse model, ACT-451840 showed the ability to cure malaria at doses of 3 × 300 mg/kg with an ED₉₀ of 13 mg/kg. In the P. falciparum SCID mouse model, it had an ED₉₀ of 3.7 mg/kg. The importance of the delivery system for ACT-451840 was shown through in vivo experiments: a 60 mg dose in corn oil was as effective as a 100 mg dose in a mixture of Tween-EtOH/water = 10:90.

ACT-451840 has shown activity against multiple parasite life cycle stages of both P. falciparum and P. vivax [104]. The MoA is suspected to be novel but is currently unknown. ACT-451840 last completed first-in-human studies in 2014 (NCT02223871) [105] and is currently awaiting a decision to proceed.