Preclinical

Once a lead compound has been identified, optimization of the structure can begin. This largely involves investigation into the structure-activity relationship (SAR) of the drug, optimising for properties such as potency (both in vitro and in vivo), solubility and metabolic stability. The candidate must also be assessed for any possible toxicity (e.g. dosing, cytotoxicity/genotoxicity levels, etc.).

M5717 (Fig. 6) was developed in 2015 by a team led by the Drug Discovery Unit (DDU) in Dundee and was shown to have potent activity against multiple stages of the Plasmodium parasite via a novel mechanism of action [78, 79].

Fig. 6

Key biological and physical properties of M5717.

Initial phenotypic screening of the Dundee protein kinase scaffold library against the 3D7 multi-drug-resistant P. falciparum strain identified a compound (M1, Fig. 7) that possessed high potency against the parasite, albeit with poor physicochemical properties. Optimization of this structure (via M2 and M3) led to improvements across the board (M5717).

Fig. 7

Key stages in the hit to lead pathway of M5717. Initial replacement of Br for F, replacement of pyridine with ethylpyrrolidine, and addition of a morpholine fragment led to the optimized compound M5717.

In addition to the nanomolar activity against the 3D7 strain, M5717 has shown almost equal potency against a number of other drug-resistant strains (K1, W2, 7G8, TM89C2A, D6 and V1/S) as well as similar potencies across multiple life cycle stages (liver schizonts, gametocytes and ookinetes).

M5717 was found to be as effective as current anti-malarial drugs (chloroquine, mefloquine, artemether, dihydroartemisinin and artesunate) when evaluated in the in vivo Plasmodium berghei mouse model for single-dose efficacy showing > 99% reduction in parasitaemia at doses of 4 × 30 mg/kg p.o. q.d. and an ED₉₀ of 0.1–0.3 mg/kg.

Due to its novel MoA (Pf eEF2 inhibition, vide infra) and its ability to clear blood-stage parasites completely, M5717 satisfies the requirements to be a long duration partner and could be used as part of combination therapy with a fast-acting compound [80]. Additionally, the compound has shown the ability to act as a transmission-blocking drug (stage IV–V) and also to be effective for chemoprotection.

In late 2017, M5717 was cleared for progression from development to Phase I clinical trials for volunteers in Australia (NCT03261401).

Identified by AstraZeneca in 2015, MMV253 (Fig. 8) is a novel triaminopyrimidine (TAP) that has shown good in vitro potency and in vivo efficacy, and acts through another novel MoA [81].

Fig. 8

Key biological and physical properties of MMV253. logD and in vivo ED₉₀ kindly provided by V. Sambandamurthy, S. Hameed P. and S. Kavanagh, personal communication, 2018.

High-throughput screening of 500,000 compounds from AstraZeneca’s library against blood-stage P. falciparum resulted in the identification of a promising series of TAPs. The initial hit (M’1, Fig. 9) suffered from hERG inhibition and poor solubility which, through lead optimization, was improved upon to give a compound that possessed high potency and desirable pharmacokinetic properties (MMV253).

Fig. 9

Key stages in the hit to lead pathway of MMV253. Initial replacement of ethylbenzene on M’1 with 2-methylpyridine resulted in lower hERG affinity and improved solubility. Substitution of the pyrrolidine in M’2 with an imidazole containing an amine spacer further improved solubility and greatly improved the potency. Addition of an N-methyl group and a cyclopropane moiety led to the optimized compound MMV253.

When screened against numerous mutant resistant strains with various mechanisms of resistance, MMV253 showed no spontaneous reduction in potency which can be attributed to its novel MoA (Pf ATP4 inhibition, vide infra). Good in vitro-in vivo correlation (IVIVC) was shown with a predicted human half-life of ∼ 36 h (which is long compared to another fast-killing drug, artemisinin, which has a human half-life of 1 hour).

As of late 2016, the pharmaceutical company Cadila Healthcare owns the license for the compound series and is now doing further lead development in order to progress the drug through preclinical trials [82].

UCT943 (Fig. 10) was identified in 2016 by a team at the University of Cape Town, South Africa and is a key compound in a novel class of 2-aminopyrazine antimalarials that has shown single dose curing ability in vivo and potential as a clinical candidate [83].

Fig. 10

Key biological and physical properties of UCT943.

The original 3,5-diaryl-2-aminopyridine series was identified from a high-throughput screen of 36608 compounds from the commercially available SoftFocus kinase library [84]. The initial hit (U1, Fig. 11) showed promising in vitro activity against the drug-sensitive NF54 strain (IC₅₀ = 49 nM) but suffered from poor solubility and high metabolic clearance. To address the poor measured in vivo stability, the labile hydroxy and methoxy groups were replaced by a single trifluoromethyl group (U2), but this change resulted in a significant loss of solubility. Significant improvements in solubility and potency were obtained by first replacing the mesyl group with a piperazine carboxamide group (U3) and subsequently introducing another nitrogen atom into the pyridine ring (UCT943) [85, 86].

Fig. 11

Key stages in the hit to lead pathway of UCT943. Initial change the phenyl substituents with a single trifluoromethyl group led to greater in vivo stability. Introduction of piperazine amide instead of methylsulfonyl and a pyrazine instead of a pyridine led to the improved solubility and potency of the optimized compound. Surprisingly, the introduction of nitrogen in the red circle resulted in complete inactivity in vivo.

In the P. berghei mouse model, UCT943 has shown the ability to cure malaria at doses of 4 × 10 mg/kg and has an ED₉₀ of 1 mg/kg. Interestingly, another closely related molecule (with nitrogen instead of carbon in the red circle) which was evaluated during the SAR study, displayed similar in vitro anti-malarial activity (IC₅₀ = 9.1 nM) and solubility (198 μM) but showed a complete lack of in vivo activity with a < 40% reduction in parasitaemia using a comparable dosage.

UCT943 is potent across multiple parasite life stages of both P. falciparum and P. vivax. Its target is Plasmodium phosphatidylinositol-4-OH kinase (Pf PI4K), which has also been implicated as the target for MMV048 (vide infra) [87]. UCT943 was in originally in place as a back-up to MMV048, however, due to preclinical toxicity, this candidate has been withdrawn.

From a discovery process by Anacor Pharmaceuticals that began in 2010 with a novel class of benzoxaborole anti-malarial compounds [88], AN13762 (Fig. 12) emerged in 2017 as the lead compound, showing excellent activity in vitro and in vivo, multi-strain efficacy and the ability to perform as a rapid-acting drug [89].

Fig. 12

Key biological and physical properties of AN13762. Solubility kindly provided by Y.-K. Zhang, personal communication, 2018.

By screening a library of boron-containing compounds (previously shown to have selective activity against fungi, bacteria, parasites and inflammation) in a whole-cell assay against P. falciparum, the initial hit compound, AN3661 (A1, Fig. 13), was identified to have potent in vitro activity against the 3D7 strain (IC₅₀ = 26 nM). Further SAR and optimization studies led to the development of A2 in which the alkyl carboxylic acid chain was moved and replaced by a substituted pyrazine ether. This compound showed greater potency but still suffered from high metabolic clearance [90]. Replacement of the ester group with an amide group (A3) led to improved metabolic stability and bioavailability, with a significant decrease in potency. Modification of the primary amide to a cyclic tertiary amide and introduction of a methyl group on the benzoxaborole gave the lead compound AN13762 which possessed improved potency and metabolic stability [89].

Fig. 13

Key stages in the hit to lead pathway of AN13762. Initial replacement of the carboxylic acid chain with pyrazine and subsequent switch of the ester to a substituted amide helped to improve in vivo stability and bioavailability leading to the optimized compound.

AN13762 has been shown to be equally potent across a wide range of drug-resistant strains. The drug has displayed similar in vivo clearance rates when compared to artesunate. There is no inherent genotoxicity (as shown in Ames assays and in vivo rat micronucleus studies), and no cytotoxicity at concentrations up to 100 μM in human cell lines [89].

The precise mechanism of action for AN13762 remains unknown, though initial MoA studies on hit compound AN3661 identified a potential target as the P. falciparum cleavage and polyadenylation specificity factor (Pf CPSF3) [91]. AN13762 has proceeded into the preclinical phase, with first-in-human studies planned for 2019 [58].

Developed in 2017 by a team at Heidelberg University, SC83288 (Fig. 14) is an amicarbalide derivative that possesses a novel chemotype for current antimalarials, may have a potentially new MoA and has shown the ability to be a fast-acting drug for the intravenous treatment of severe malaria [92].

Fig. 14

Key biological and physical properties of SC83288. In vivo ED₉₀ kindly provided by M. Lanzer, personal communication, 2018.

An in silico screen of a library of small molecule compounds for their ability to dock into P. falciparum lactate dehydrogenase led to the identification of amicarbalide (S1, Fig. 15), which was found to be highly potent (IC₅₀ = 10 nM) against the Dd2 strain [93]. In order to overcome a potential DNA binding effect, an amidine group was replaced with a sulfonamide linker leading to S2 which possessed better solubility and improved metabolic stability. Further modification of the other amidine group with a substituted piperazine ring (S3) led to improved potency and water solubility, but the compound suffered from poor permeability. Ultimately, replacement of the butyl chain with an acetyl group led to the highly potent lead compound SC83288.

Fig. 15

Key compounds in the discovery of SC83288. Initial modification of one amidino group with a sulfonamide linker (S2) resulted in improved solubility. Further modification of the remaining amidine group with substituted piperazine moieties ultimately led to the optimized compound with good solubility, permeability and high potency.

SC83288 has been shown to be potent against a number of drug-resistant strains at IC₅₀ values of < 20 nM and is also efficacious against early-stage (I–III) gametocytes (IC₅₀ = 199 nM) but not against late-stage gametocytes (IV and V). It has an excellent safety profile, with no cytotoxicity, genotoxicity or hERG binding. In the Plasmodium vinckei rodent malaria model, SC83288 was able to fully cure the infection at a dose of 4 × 20 mg/kg i.p. q.d. with no resurgence of infection. It is, however, completely inactive against P. berghei.

While the exact MoA of SC83288 is unknown, the generation of resistant clones has identified Pf ATP6 as a possibly relevant target. However, it has been shown that SC83288 does not directly inhibit this target suggesting Pf ATP6 may have a less direct role in the mechanism of SC83288. Pf MDR2 has been identified as another possible mechanism of resistance, facilitating the clearance of the drug from the parasite. SC83288 has been evaluated against artemisinins, showing no cross-resistance and highlighting its potential as an alternative to artesunate for the treatment of severe malaria when combined with a slow-acting partner drug [94].

Discovered in 2010 by a team at Portland State University and further developed by DesignMedix, DM1157 (Fig. 16) is part of a class of compounds known as “reversed chloroquines” (RCQs), designed to overcome chloroquine-resistant and chloroquine-sensitive strains of the malaria parasite. The compound has been shown to be efficacious in vitro and in vivo [95].

Fig. 16

Key biological and physical properties of DM1157. CLint HLM and in vivo ED₉₀ kindly provided by D. Peyton, personal communication, 2018.

CQ resistance is known to be linked to the P. falciparum chloroquine resistance transporter (Pf CRT): mutations to this target facilitate the expulsion of CQ from the parasite. A class of molecules has been identified, so-called “reversal agents”, that can inhibit Pf CRT, thus slowing the exportation of CQ from the parasite. By combining the chloroquinoline core of CQ (D1, Fig. 17) with the known reversal agent, imipramine, the first RCQ (D2) was designed, but this molecule suffered from poor bioavailability and metabolic stability [96]. Subsequent SAR studies resulted in the substitution of the imipramine motif with a 1-(2,2-diphenylethyl)piperazine moiety which led to a compound (D3) that was more stable to metabolic cleavage, but suffered from a high cLogP. In order to overcome this, the two phenyl groups were replaced with pyridines and the piperazine replaced with an aminopiperidine resulting in the lead compound, DM1157 that possessed a lower cLogP value (3.6) while still maintaining high potency against both CQ-resistant (Dd2 = 1.6 nM) and CQ sensitive strains (D6 = 0.9 nM) when compared to CQ (cLogP = 5.1, Dd2 = 102 nM, D6 = 6.9 nM).

Fig. 17

Key compounds in the discovery of DM1157. The initial combination of the reversal agent, imipramine, with the CQ core resulted in the potent RCQ compound D2. Subsequent replacement of the reversal agent with 1-(2,2-diphenylethyl)piperazine (D3) and further modification with pyridine rings led to improved potency and cLogP values for the optimized compound.

In a P. berghei rodent model, DM1157 showed good efficacy both orally and subcutaneously. Most notably, a > 99.9% reduction in parasitaemia was seen at an oral dose of 4 × 30 mg/kg with 2/3 mice cured 30 days post-infection. DM1157 has also been evaluated against P. falciparum and P. vivax multi-drug resistant field isolates in Indonesia and was found to be threefold more potent than CQ in both species [97].

CQ is known to bind to heme and inhibit β-haemozoin formation. DM1157 (and other RCQ compounds) have been shown also to act in this manner, but with much higher levels of inhibition of β-haemozoin both in vitro and in vivo. DM1157 is currently in Phase I trials to evaluate its safety and pharmacokinetics in humans (NCT03490162).