Substituted Aminoacetamides as Novel Leads for Malaria Treatment

Abstract Herein we describe the optimization of a phenotypic hit against Plasmodium falciparum based on an aminoacetamide scaffold. This led to N‐(3‐chloro‐4‐fluorophenyl)‐2‐methyl‐2‐{[4‐methyl‐3‐(morpholinosulfonyl)phenyl]amino}propanamide (compound 28) with low‐nanomolar activity against the intraerythrocytic stages of the malaria parasite, and which was found to be inactive in a mammalian cell counter‐screen up to 25 μm. Inhibition of gametes in the dual gamete activation assay suggests that this family of compounds may also have transmission blocking capabilities. Whilst we were unable to optimize the aqueous solubility and microsomal stability to a point at which the aminoacetamides would be suitable for in vivo pharmacokinetic and efficacy studies, compound 28 displayed excellent antimalarial potency and selectivity; it could therefore serve as a suitable chemical tool for drug target identification.


Introduction
Malaria is ad evastating parasitic disease, which is endemici n many parts of the developing world. Human malaria is caused by five species of Plasmodium,o fw hich, P. falciparum causes the greatest number of deaths, affecting many parts of Africa. P. vivax causes severemorbidity andaffects mainly Latin America and South East Asia. [1] In 2017, there were 219 million reportedc ases of malaria,a nd > 400 000 deaths, many of which were in children under the age of five years. [2] The malaria parasite is transmitted to humans throught he bite of an infected female anopheles mosquito when takingablood meal. Following infection, malaria parasitesi nt he form of sporozoites, migrate from the skin to the liver,where they invadeand develop into merozoites.W hen merozoites outgrow liver cells, they are released into the bloods tream, invading red bloodc ells, where they continuet og rowa nd develop, repeating the cycle in the erythrocytes. During the intraerythrocytic stages of infection, some parasites differentiate into the sexual form (gametocytes), while others remaina st he asexual form, progressing through multiple stages of development within the blood cells. When the parasites are mature,i nfected red blood cells burst, releasingp arasitesi nto the bloodstream, causing the febrile symptoms associatedw ith malaria. Asexual merozoites are then free to invadeo ther red blood cells, whereas gametocytes may be taken up through the bite of af eeding mosquito and transmitted to another human, perpetuating the malaria parasite life cycle. [3] Current front-line therapies for the treatment of malaria are failing due to the increasing development of drug resistance and new antimalarial treatments are urgently needed. [4] Furthermore, novel therapies that targett he malaria parasite in all life-cycle stagesa re required, to both treat Herein we describe the optimization of ap henotypich it against Plasmodium falciparum based on an aminoacetamide scaffold.T his led to N-(3-chloro-4-fluorophenyl)-2-methyl-2-{[4methyl-3-(morpholinosulfonyl)phenyl]amino}propanamide (compound 28)w ithl ow-nanomolar activity against the intraerythrocytic stages of the malaria parasite, and which was found to be inactivei namammalian cell counter-screen up to 25 mm.I nhibition of gametesi nt he dual gamete activation assay suggests that this family of compounds may also have transmission blocking capabilities. Whilst we were unable to optimize the aqueous solubility and microsomals tability to a point at which the aminoacetamides would be suitable for in vivo pharmacokinetic and efficacy studies, compound 28 displayed excellent antimalarial potencya nd selectivity;i t could therefore serve as as uitable chemical toolf or drug target identification. and preventt he spread of malaria and to help in the process of eliminating this disease. [5] Results and Discussion

Project initiation
To develop potentialn ew antimalarials, we focused our attention on the publicly available resultso fahigh-throughput screening (HTS) campaign of nearly two million compounds carried out by GlaxoSmithKline (GSK), to identify new chemical matter with antimalarial activity. [6] The screening campaign identified > 13 500 compounds malaria blood-stage actives (Tres Cantos Antimalarial Set, TCAMS). Chemicalf iltering and prioritizationr esulted in 47 different chemotypesw ith antimalarial potency (EC 50 ) < 2 mm.H erein we describe the selection of one particular series of inhibitors from the TCAMS library screen and the subsequento ptimization of an ew class of antimalarials, based on as ubstituted amidoacetamide core.

Hit selection
Amidoacetamide TCMDC-123553 (Series 10) [7] was selected as a new chemical startingpoint based on good antimalarial potency against chloroquine/pyrimethamine-sensitive and -resistant strains,s uitable physicochemical properties for furtherd evelopment [8] and was not subjectt od rug discovery efforts from any other MMV funded programs. Series 10 comprised ac luster of two compounds:T CMDC-123553 and TCMDC-125117 ( Figure 1). The enamide moiety of TCMDC-125117 was flagged as ap ossible Michael acceptor and with the added risk of epoxidation of the double bond to give ar eactive intermediate. [9] Therefore, our focus was directed towardT CMDC-123553 (1) for further in vitro assessment.
As as uitable chemical startingp oint, TCMDC-123553 (1)d isplayed good antimalarial activity in vitro and was inactive in a mammalian counter-screen. In ap reliminary malaria parasite rate of kill assay (the parasite reduction ratio (PRR) assay [10] ), 1 showedarelativelys low rate of kill, intermediate between pyrimethamine and atovaquone ( Figure 2). In this assay,p arasites were treated with 10 EC 50 concentrations of 1 and samples of parasitest aken from treated cultures after 24 and 48 h, and the number of viable parasites determined.
Compound 1 wasinvestigated for activity againsto ther Plasmodium life-cycle stages. In a P. berghei liver-stages chizont assay, [11] compound 1 was found to be inactive up to 50 mm.I t displayed no effect in P. cynomolgi liver-stage assay [12] against both large forms and small forms (the latter indicative of P. vivax hypnozoites)a t1 0mm.T herefore, compounds of this series do not appear to be suitable for malaria chemoprotection or radicalc ure. Compound 1 (incubated at ac oncentration of 1 mm)g ave decreases of 99 and 81 %i nm ale and female gamete formation( sexual stages found in the mosquito) assays, [13] respectively.H owever, although 1 was an inhibitor of male and female gamete formation,i tw as inactive in a late-stage (stage IV/V) gametocyte assay [14] at 40 mm.T his latter assay measures the ability of compounds to kill mature gametocytes. Based on the inhibition in the dual gametef ormation assay the aminoacetamide series would be expected to show transmission blocking activity,b ut this would need to be confirmed in as tandard membrane feeding assay,w hich uses mosquito-based readouts.

Optimization of R 1
Modifications at R 1 (Figure 3a nd Ta ble1)w ere initially investigated around the substituted phenyl ring. Modificationsw ere focused on improving or retaining activity,w hilst increasing metabolic stability and solubility.W ee nvisaged that an increasei nm etabolic stability could be achieved by modifications to the phenyl ring by the introduction of heteroatomst o both modulate logP and remove potential sites of metabolism. Alternatively,r eplacement of the phenyl ring with aliphatic polar and/orb asic groups, could simultaneously improvem et-   (3) gave a1 0-foldd ecrease in activity.F urthermore, removalo f one or both halogensh ad little effecto nm etabolic stability. Synthetic modifications at R 1 were then directed towarda romatic heterocycles. Direct replacement of the di-halogenated phenylo f1 with ap yrazine (5)d id not improve solubility or metabolic stability and lost all antimalarial activity. Thiazole (6) and oxazole (7)h owever,d id improves olubility although all antimalarial activity was lost and metabolic stabilityw as not improved.
Despite the relatively low clogP,t he compounds still showed poor metabolic stability. We then focusedo na liphatic heterocycles. Substitutingt he R 1 di-halogenated anilineo f1 for morpholine 8,l ost all antimalarial activity,a lthough led to an improvement in metabolic stability. Cyclohexylamine 9 retained some activity,b ut suffered from % 20-foldd rop in antimalarial potencyr elative to compound 1,a nd was metabolically unstable. The difluoro derivative (10)s howeds imilar activity to compound 9,b ut with significantly improved solubility,p ossibly due to the difluorog iving as mall dipole moment. [16] Replacement of the morpholine oxygen atom in 8 with difluoro 11 was also inactive, and lost metabolic stability relative to 8.T he addition of anitrile group on the cyclohexyl ring of 9,asexemplified by 12,l ed to al oss of activity.R eplacement of the R 1 amino cyclohexyl moiety of 9 with ap iperazine (13)i mproved metabolic stability and aqueous solubility but not metabolic stabilitya nd also lost all antimalarial activity.S ubstitution at R 1 with piperidine 14 afforded ac ompound with lower solubility and microsomal stability than 13,and again rendered the compound inactive. Efforts to improve potency and physicochemical properties were then directed toward changes at the R 2 position.  Substitution of the methyl group of 1 at R 2 with fluorine to afford 16,d isplayed as mall drop in potency, but 16 was still rapidly metabolized ( Table 2). Replacement of the methyl group on R 2 with ac hlorine atom (15), displayed am oderate improvement in potency, but aqueous solubility and metabolic stabilityw ere still poor.R emoval of the methyl group altogether with 17,d id not improvem etabolic stability or solubility and gave rise to a % 100-fold dropi np otency. Modificationsa t R 2 were then directed toward replacement of the morpholine sulfonamide moiety.R emoval of the morpholine oxygen atom from 1 gave the piperidines ulfonamide 18,w hich showed similar potencyb ut increased lipophilicity and decreased aqueous solubility.T he smaller and less lipophilic diethyl sulfonamide (19)a nd dimethyl sulfonamide (20)d id not improve metabolic stabilityo rs olubility relative to 1,a lthough 19 retained potency, and 20 only showed as mall drop. Replacement of the sulfonamide morpholine group of 1 with am orpholine amide (21), led to al osso fa ctivity.H owever,c ompound 21 showedi ncreased solubility,a lthough the metabolic stability was still too high. To try and recover potency,w er emoved the carbonyl group of morpholine amide 21 to afford benzyl morpholine 22.W ee nvisaged that free rotationo ft he alkyl-linked morpholine aroundt he phenyl ring of R 2 may allow the morpholine group to find an optimal interaction with the unknown biological target. However, 22 was inactive against malaria parasites.Wethen explored the bridged morpholine sulfonamidem oiety 23,t od ecrease the planarity of the molecule, with av iew to increasing solubility through the possible disruption of crystal packing [17] Compound 23 retained only a slightly reduced potencyr elative to compound 1,a nd may have shown an marginal improvement in solubility but was very unstablew ith microsomes. We then movedt ot he, Increasing antimalarial activity:m odifications to the linker Initial changes to the aliphatic linker soughtt od etermine the importance of the linker hydrogen bond donors for antimalarial activity,t hrough the systematic methylation of each NH functionality.M ethylation of the R 1 amide NH (25)g ave rise to al oss in potency.H owever,m ethylation of the R 2 aniline NH (26)w as tolerated although aqueous solubility and metabolic stabilityw ere not improved compared with 1.R emoval of the amide carbonyl to afford 27 resulted in > 100-fold drop in potency and also decreased aqueouss olubility.W et hen investigated if the metabolici nstability of 1 was associated with metabolism a to the carbonyl, which would also be N-dealkylation, ak nown phase1 biotransformation. [18] Ac ommon approach to decrease metabolic instability is the geminal dimethylationo fm etabolically labile regions. [19] The dimethylated analogue 28 did not show improved metabolic stabilityb ut displayed an approximate 20-fold improvement in potency relative to hit compound 1 (Table 3).

Met ID studies
To understand what was causing the microsomal instability,w e soughtt oi dentify the chemicalf unctional groups that were susceptible to metabolism,i ncubating mousel iver microsomes in the presence 1 andm onitoring for metabolites by mass spectrometry. Combining key SAR Af inal attemptw as made to investigate different combinations of previously tried functional groups, to improvep hysicochemicalcharacteristics as well as potencya gainst P. falciparum ( Table 4). Substitution of the linker moiety with a geminal dimethyl group, combined with bridged morpholine analogues at R 2 ,a fforded 29 and 32.B oth gave a1 0-fold increase in potency relative to 1.H owever,s olubility and metabolic stability were not improved. Newc ombinations at R 2 ,r eplacingt he methyl group with methoxy as exemplified by 30,also resulted in a1 0-fold increase in potencya nd improved solubility relative to 1,a lthougha gain, metabolic stabilityw as not improved. Substitution of the R 2 morpholine on 1 with the spirocyclic morpholine sulfonamide of 31 displayed similarp otency to 1 but the desired increased levels of metabolic stabilitya nd aqueous solubility were not obtained.

Conclusions
Through iterative rounds of drug design ands ynthesis, we were able to improvet he antimalarial potency of 1 by > 10fold to afford 28,w hich displayed single digit nanomolar potency against P. falciparum andg ood cellular selectivity.I t should be noted, however,t hat not all compounds in this series inhibited growth of P. falciparum to 100 %i nvitro. Unfortunately,w ew ere unable to identify compounds that combined both potency and good metabolic stabilityt op rogress to in vivo pharmacokinetic studies. Furthermore, we weren ot able to understand the reasons for the metabolic instability. Within this series, only compounds with very low lipophilicity (clogP < 1) displayed the desired levels of microsomal stability (< 5mLmin À1 (g liver) À1 ). Unfortunately,t hese compounds lost all antiparasitic activity.T herefore, during the optimization of the aminoacetamides, lowering lipophilicity was not sufficient to significantly decrease microsomal instability to the required levels.I ts hould also be noted that compounds with very low logP values are likely to have al arge free unbound fraction, which could lead to higher levels of clearance in vivo. [19]  Given the excellent antimalarial activity and selectivity of 28, mode of action studies to determine the molecular target of this series may be valuable. These results may open new avenues to identify novel chemotypesw ith improved metabolic stability, aqueous solubility and potent antimalarial activity.

Experimental Section
Please refer to the Supporting Information for all experimental details.
The human biological samples were sourced ethically,a nd their use in research was in accord with the terms of the informed consents under an IRB/EC-approved protocol.