In vitro and in vivo evaluation of the antimalarial MMV665831 and structural analogs
Antimalarial candidates possessing novel mechanisms of action are needed to control drug resistant Plasmodium falciparum. We were drawn to Malaria Box compound 1 (MMV665831) by virtue of its excellent in vitro potency, and twelve analogs were prepared to probe its structure-activity relationship. Modulation of the diethyl amino group was fruitful, producing compound 25, which was twice as potent as 1 against cultured parasites. Efforts were made to modify the phenolic Mannich base functionality of 1, to prevent formation of a reactive quinone methide. Homologated analog 28 had reduced potency relative to 1, but still inhibited growth with EC50 \xe2\x89\xa4 200 nM. Thus, the antimalarial activity of 1 does not derive from quinone methide formation. Chemical stability studies on dimethyl analog 2 showed remarkable hydrolytic stability of both the phenolic Mannich base and ethyl ester moieties, and 1 was evaluated for in vivo efficacy in P. berghei-infected mice (40 mg/kg, oral). Unfortunately, no reduction in parasitemia was seen relative to control. These results are discussed in the context of measured plasma and hepatocyte stabilities, with reference to structurally-related, orally-efficacious antimalarials.
<p>Malaria remains a deadly disease, leading to approximately 228 million malaria cases worldwide and killing over 400,000 in 2018.1 Artemisinin combination therapies have contributed in large part to a decline in malaria mortality over the last 15 years; however, resistance to these treatments is emerging, and the estimated malaria caseload has been on the rise in the last two years. Thus, the need for novel antimalarial compounds remains urgent.1, 2 Malaria symptoms in humans are caused by the blood-stage growth of Plasmodium spp., of which P. falciparum is responsible for ~96% of malaria cases worldwide. To accelerate the development of novel antimalarial drugs, a collection known as the "Malaria Box" was assembled from over 20,000 compounds with confirmed in vitro activity against blood-stage P. falciparum, and low general cytotoxicity.3 From the 20,000 hits, 400 compounds were selected using several criteria, including commercial availability, oral bioavailability, and chemical diversity. This final set contained 200 "drug-like" and 200 "probe-like" compounds, classified according to their perceived druglikeness.</p><p>One of us (Klemba) identified compound 1 (Figure 1, MMV665831) as having excellent in vitro growth inhibition of asexual blood-stage P. falciparum, and similar high potency in inhibiting amino acid efflux4 from parasitized erythrocytes.5 We have previously observed such behavior in a study of mefloquine (MQ) and its non-piperidine analogs, and suggested that the antimalarial action of MQ may stem from inhibition of hemoglobin endocytosis.2 Repeat assay with purchased authentic samples of 1 and its N,N-dimethyl analog 2 (ChemDiv) confirmed these activities. Thus, we sought to explore the structural determinants of antimalarial action of 1 and 2. In particular, we wanted to establish whether potency could be sustained when the phenolic Mannich base functionality (a likely trigger of the probe-like designation6) and C3-carboxylic ester were converted to less-reactive moieties (Figure 1).</p><p>To enable the synthesis of such analogs of 1/2, we developed a gram-scale synthesis of the core 7-hydroxybenzothiophene 3, which serves as a Mannich reaction precursor for 1 and its analogs. (Scheme 1) A facile Gewald reaction from cyclohexanone, sulfur, and ethyl cyanoacetate7 constructed the known thiophene 4 in 73% yield, which was benzoylated to give 5 in 95% yield. Regioselective oxidation at C7 was achieved with CuSO4/potassium persulfate8 to give 6 with 58% yield.</p><p>To achieve the conversion of ketone 6 to the desired phenol 3, we employed an α-halogenation/dehydrohalogenation sequence. Whereas bromination of 6 with Br2 or NBS resulted in a mixture of mono- and di-brominated product, regioselective monobromination was achieved with Bu4NBr39 affording 7 in 82% yield. Treatment of 7 with lithium carbonate in DMF at 120 °C for 2 h cleanly eliminated HBr;10 addition of water precipitated phenol 3 in 94% yield, which was sufficiently pure for further steps.</p><p>Since the 2-ethyl ester in 1 was viewed as a potential hydrolytic liability in vivo, we desired to prepare and evaluate amide analogs. Compound 3 dissolved poorly in ethanolic methylamine (33 wt%) at 25 °C, and no conversion was observed after 24 h; the mixture was heated to 60 °C in a sealed tube overnight, but instead of the ethyl ester 8, amine 9 was isolated in 45% yield due to aminolysis of the benzamide (Scheme 2). The silyl ether 10 was prepared to improve substrate solubility, and subsequent aminolysis at 35 °C gave full conversion. Under these conditions the desired methyl amide 11 was isolated in 40% yield, together with 42% of debenzoylated byproduct 12. The factors contributing to the stability of the ester group towards aminolysis remain unclear, but byproduct 12 proved useful as shown below. Silyl ether 11 was easily deprotected with TBAF to give phenol Mannich precursor 8 in 88% yield.</p><p>To provide isosteric comparison with the methyl amide 8, the methyl ester 13 was prepared from methyl cyanoacetate, using the method for 3 (Scheme 3). The byproduct 2-aminobenzothiophene 12 obtained from the aminolysis of 10 was acetylated with 4-chlorobenzoyl chloride to give 14, which was desilylated to give phenol Mannich precursor 15. Curiously, the reaction of 12 with 4-methoxybenzoyl chloride, even with 4-DMAP catalysis, did not give any conversion. Fortunately, thiophene 4 could be benzoylated with the 4-methoxybenzoyl chloride to give 16, which was converted to precursor 17 (Scheme 3).</p><p>Phenols 3, 8, 13, 15, and 17 readily underwent Mannich reaction with a series of secondary amines in the presence of formalin, giving the desired products 1, 19-26 in 44–92% yield. (Table 2) Reaction of 3 with the 1° amine 1-adamantylamine and formalin initially gave hemiaminal ether 18; treatment with aqueous acid gave the desired product 27.</p><p>The P. falciparum growth inhibition potencies of compound 1, 19-27 were tested in a 48-hour growth assay. Since the 3D7 strain of the parasite was used to select compounds for the Malaria Box,3 we employed it as well; MQ was used as positive control. Within error compound 1 is equipotent with MQ, with a P. falciparum growth inhibition EC50 values of 27 ± 8 and 16 ± 6 nM, respectively. (Entries 1 and 2, Table 2). Our limited series modifying the para-position of the benzamide (Y, Table 2, entries 2, 4, 5) shows Y = H (1, 27 ± 8 nM) is favorable to Y = Cl (19, 70 ± 18 nM), although Y = OCH3 (20, 66 ± 35 nM) is indistinguishable from Y = H (1). Similarly, our limited modification of the 3-carboxy substituent (X) indicates that an amide is not tolerated (22, Table 2, Entry 7, > 1,000 nM). Methyl ester 21 is equipotent with 1, within error (Entry 6, Table 2, 53 ± 25 nM). Thus, the poor potency of the methyl amide 22 is not steric in origin.</p><p>To our delight, varying the NR1R2 moiety is well-tolerated, and can result in improved potency. Whereas dimethylamine analog 2 is equipotent to 1 (Entry 3, Table 2, 24 ± 8 nM); dipropylamine analog 23 and pyrrolidine analog 24 appear slightly more potent than 1 (Entries 8 and 9, Table 2, 16 ± 2, 16 ± 5 nM, respectively); piperidine analog 25 is the most potent in this series (Entry 10, Table 2, 13 ± 2 nM). Interestingly, the isosteric morpholine analog 26 is 7- to 8-fold less potent than piperidine analog 25 (Entry 11, Table 2, 100 ± 50 nM). It is tempting to speculate that the reduced basicity of morpholine related to piperidine may drive this reduced potency.11 Compound 27 with its 1-adamantyl (1-Ad) group was assessed to probe the effect of increased lipophilicity in the high potency of 25. The significant (14-fold) loss of potency compared to 1 (Entry 12, Table 2, 388 ± 116 nM), may well be a consequence of the significant steric bulk of the 1-Ad group.</p><p>Compound 1 is classified as a "probe-like" member of the Malaria Box, but no specific explanation for this designation was given in the original publication.3 As mentioned above, we suspected the phenolic Mannich base functionality of 1 was to blame, and application of the PAINs (Pan Assay Interference Compounds) filter6 in SwissADME12 did generate an alert for this functionality.</p><p>Elimination of the phenolic Mannich base moiety to a highly electrophilic quinone methide13,14 and subsequent reaction with biological nucleophiles15–17 may be responsible for promiscuous binding sometimes exhibited by this class of compounds (Figure 2). More importantly, formation of this electrophile in vivo could lead to idiosyncratic toxicity.</p><p>To block this potential reactivity, we proposed two different analogs: a) one resulting from insertion of an additional methylene i.e., compound 28, and b) the other resulting from methylation of the phenol, i.e., compound 29.</p><p>To synthesize the desired homologated analog, phenol 3 was formylated selectively at the ortho- position with paraformaldehyde and MgCl2.18,19 (Scheme 4) However, attempted Henry reaction of 30 was unsuccessful, presumably due to its poor solubility in nitromethane. We thus prepared the silyl ether 31 to improve solubility as well as reduce polarity of the products. Henry reaction of silyl ether 31 catalyzed by AcOH and pyridine20 was successful, affording the desired product 32 in 56% yield.21 The nitrostyrene 32 was reduced with sodium borohydride at 0 °C to give 33,22 and the silyl ether was deprotected with TBAF to give nitro alkane 34 in 83% yield. The nitro alkane 34 was reduced under catalytic hydrogenation conditions, and a subsequent Eschweiler-Clarke reaction gave the homolog 28. Unfortunately, insertion of the additional methylene in 28 caused an approximate 7-fold loss of potency compared to 2 (Entry 13, Table 2, 182 ± 72 nM). However, the fact that 28 retained significant growth inhibition potency strongly suggests that quinone methide formation is not the basis of the antimalarial activity of this compound class.</p><p>The o-formyl phenol 30 was readily methylated using methyl iodide/K2CO3, giving 35 in 81% yield (Scheme 5). Interestingly, the standard reductive amination protocol (sodium triacetoxyborohydride in DCE23) was not effective for reaction of 35 with diethylamine. Fortunately the alternative Ti(OiPr)4/NaBH3CN protocol24 recommended by Abdel-Magid et al.23 did afford 29 in 69% yield, with 25% recovered starting material 35. Unfortunately, methylation of the phenol completely abrogated potency (Entry 14, Table 2, EC50 > 1,000 nM).</p><p>Since structural modifications to prevent quinone methide formation from 1 or 2 significantly reduced potency, we thought it would be worthwhile to investigate the thermal stability of these compounds. After all, two WHO-approved and several investigational antimalarials25–29 contain the phenolic Mannich base functionality (Figure 3). To our knowledge, formation of quinone methide intermediates has not been implicated for any of the phenolic Mannich base antimalarials in Figure 3, or others reported to have in vitro activity.30,31 But studies of phenolic Mannich bases directed at other diseases/targets have demonstrated quinone methide formation in compounds ranging from quite reactive14–17 to fairly robust.13,32 Thus we subjected 2 to a series of tests (Scheme 6). Heating 2•HCl in methanolic diethylamine at 40 °C for 2 days did not result in any observable change by 1H NMR or HRMS. Not only did the dimethylamino group remain intact, so did the ethyl ester, consistent with our observation of the low reactivity of 3 towards amidation (Scheme 2). Compound 2•HCl also proved stable in D2O at 50 °C after 7 days, but heating at 90 °C overnight resulted in the formation of a precipitate, which was insoluble in D2O or CDCl3. This result suggests the quinone methide intermediate did form under neutral conditions at 90 °C, and subsequently polymerized. However under acidic conditions (0.1 M DCl in D2O), 2•HCl was quite stable after 1 day at 90 °C, only undergoing H-D exchange selectively at C4, forming 45.</p><p>Thus, the ethyl ester moiety of 2 (and by extension 1) is remarkably stable to acidic hydrolysis. Together these experiments indicated that 1 might be stable enough to exert antimalarial activity in vivo, and thus we tested 1 (40 mg/kg po) in a mouse model of blood-stage malaria (P. berghei). Unfortunately, compound 1 did not reduce parasitemia in vivo compared to vehicle control (See Figures S1, S2 in Supplementary Material). Plasma (mouse) stability studies of 1 showed no loss of parent over 120 minutes, indicating that the ethyl ester moiety was quite stable towards plasma carboxylesterases. Mouse hepatocyte stability experiments showed rapid loss of parent with a t1/2 of 11 min. However, a number of other phenolic Mannich Base antimalarials have similar rates of liver metabolism, and yet retain in vivo efficacy (e.g. 36, 39-44, see Supplementary Material, Table S3).26–29 Without performing oral pharmacokinetics and identifying metabolites (and their antimalarial efficacy), it is impossible to know whether the failed efficacy of 1 is due to poor plasma exposure, or to a (rare) species difference (P. berghei vs P. falciparum) in drug sensitivity.33,34</p><p>In conclusion, we prepared a series of analogs of 1 featuring modifications at the C2-benzamide, C3-ester, C6-Mannich base, and C7-phenol moieties. In vitro P. falciparum growth inhibition potencies revealed that neither an electron-withdrawing group or electron-donating group on the C2-benzamide improved potency (cf. 19, 20 with 1), that an amide cannot substitute for an ester at the C3 position (cf. 22 with 1, 21), and that the C7-phenol is essential for potency (cf. 29, 1). Increasing lipophilicity of the NR2 unit of the C6 Mannich base can double potency (cf. 23-25 with 1,2), but steric constraints eventually limit this effect (cf. 27 with 1,2). Attempts to block quinone methide formation by homologation of 1 to 28 reduced, but did not eliminate antimalarial potency (cf. Table 2). Thus, antimalarial action of 1 does not appear to require formation of the quinone methide. In fact, the Mannich base and ester functionalities of 2 (and by extension 1) were found to be quite stable under neutral and acidic conditions, and no loss of 1 was seen in mouse plasma after 2 h. Unfortunately, 1 was rapidly consumed in mouse hepatocytes, and it failed to show oral efficacy at 40 mg/kg in a mouse model of malaria.</p>