Antimalarial N¹,N³-Dialkyldioxonaphthoimidazoliums: Synthesis, Biological Activity, and Structure–activity Relationships

Abstract

Here we report the nanomolar potencies of N¹,N³-dialkyldioxonaphthoimidazoliums against asexual forms of sensitive and resistant Plasmodium falciparum. Activity was dependent on the presence of the fused quinone-imidazolium entity and lipophilicity imparted by the N¹/N³ alkyl residues on the scaffold. Gametocytocidal activity was also detected, with most members active at IC₅₀ < 1 μM. A representative analog with good solubility, limited PAMPA permeability, and microsomal stability demonstrated oral efficacy on a humanized mouse model of P. falciparum.

Substantial progress has been made in decreasing the global burden of malaria over the past decade. Worldwide, 20 million fewer cases of malaria were recorded in 2017 as compared to 2010.¹ Deaths due to malaria have declined, from 607, 000 in 2010 to 435, 000 in 2017.¹ Furthermore, low attrition rates among recent clinical trial candidates hint at a potentially rich antimalarial drug pipeline in the offing.² However, optimism engendered by these encouraging statistics belies two worrying trends. First, several high burden countries on the African continent have seen a resurgence in malaria in spite of vigorous efforts to curtail spread.¹ Second, the Greater Mekong Region has witnessed intensification of the multidrug-resistant Plasmodium falciparum colineage KEL1/PLA1.³ These events highlight the fragility of gains made against malaria and the need for constant vigilance if they are to be sustained. Like other infectious diseases, the emergence of resistant parasite strains is a question of when and not if. Regardless of the prevailing optimism invoked by the current status of the drug pipeline, there is a constant need for new agents to protect vulnerable populations, prevent relapses of vivax infection, and disrupt disease transmission.² Eradication of malaria, which is a key Millennium Development Goal set by the United Nations, is achieved only by breaking disease transmission.⁴ Unlike malaria control which treats the symptoms caused by asexual parasites, eradication focuses on eliminating the sexual forms (gametocytes) that are directly responsible for infecting the anopheline vector and perpetuating the plasmodial life cycle. Ideally, antimalarial leads should target both asexual and sexual intraerythrocytic parasites with comparable potencies.⁵

In an effort to identify lead compounds with potent activity against both life-cycle stages of P. falciparum, Tanaka and co-workers reported the demethylation of the diaminonaphthoquinone SJ000030570 to the monomethylamino analog SJ0000541602 on prolonged exposure to light, followed by cyclization of the latter to the dioxonaphthoimidazolium SJ0000561981 (Figure 1A).⁶ Both SJ0000541602 and SJ0000561981 were photostable and exceptionally potent against asexual ring stages and late stage (stages III–V) gametocytes (Figure 1A).⁶

Figure 1.

(A) Stepwise conversion of SJ000030570 to SJ0000541602, and SJ0000561981 and their EC₅₀ values against the asexual and sexual stages of P. falciparum 3D7.⁶ Structures of (B) YM155 and (C) analogs 1–4⁷ and their IC₅₀ values (±SD) against asexual stages of drug sensitive NF54 and drug resistant Dd2 P. falciparum. Parasitemia was assessed by the parasite lactate dehydrogenase assay. Positive controls were chloroquine (IC_50NF54 = 8.2 ± 0.4 nM; IC_50Dd2 = 118 ± 3 nM) and artesunate (IC_50NF54 < 5 nM; IC_50Dd2 = 18.8 ± 1.5 nM).

The dioxonaphthoimidazolium scaffold of SJ0000561981 is present in YM155 (sepantronium bromide, Figure 1B), an antitumor agent and suppressor of the antiapoptotic protein survivin.⁸ This prompted us to ask if YM155 could emulate the antimalarial activity of SJ0000561981 and more importantly, if the scaffold is an incipient antimalarial pharmacophore. Hence, YM155 and several analogs (1–4) that were previously investigated on human cancer cell lines⁷ were screened against the erythrocytic stages of drug sensitive and drug resistant P. falciparum (Figure 1B). Disappointingly, YM155 displayed only modest activity, but its analogs 1 and 4 proved more potent. We were particularly intrigued by 4 whose outstanding activity is ostensibly linked to the absence of cyclized entities at the ring nitrogens N¹/N³. This led us to propose modifying the alkyl substituents in 4 by homologation or branching as a means of optimizing the antimalarial potential of the scaffold. To this end, a series of N¹,N³-dialkyldioxonaphthoimidazoliums (4–35) were synthesized and evaluated against the asexual and sexual intraerythrocytic stages of P. falciparum.

Briefly, our target compounds were obtained by introducing alkyl groups and their branched/cyclized variants at one of the imidazolium N atoms while retaining an unchanged alkyl at the other N atom. For instance, with methyl at N¹, we inserted ethyl, propyl, butyl, isopropyl, or cyclopropyl in turn at N³ and this was repeated with each of the aforementioned N³ substituents at N¹. In this way, series A (4–24) was prepared and evaluated on the asexual stages of P. falciparum. The results prompted the synthesis of two additional series, namely, the nonquaternized series B (36–40) in which only N¹ was substituted and series C (25–35) which has isobutyl at N¹, the same groups at N³ as in series A in addition to longer alkyl side chains (isobutyl, n-hexyl, n-octyl, n-decyl, n-dodecyl) (Scheme 1). Several members in series A–C have been reported in the literature, namely, 4, 10, 19;⁹5, 7, 9, 11, 14, 16, 25, 36–40;¹⁰24, 31;¹¹12, 13,¹² and 15.¹³ We followed the method of Ho et al.⁷ to obtain series A–C.

Scheme 1.

Reagents and conditions: (a) R¹-amine, EtOH, rt, 18 h; (b) acetic anhydride, conc. H₂SO₄, rt, 1.5 h; (c) R²-amine, toluene, 45 °C, 4 h; (d) 48% HBr (aq), EtOH + EtOAc, 45 °C, 4 h to rt overnight; (e) NH₃ in MeOH, EtOH, 35 °C, 3 h; (f) 48% HBr (aq), 2 M NaOH (2 drops), EtOH, 50 °C, 1 h.

The growth inhibitory IC₅₀ values of series A compounds on the asexual forms of Dd2 (drug resistant) and 3D7 (drug sensitive) strains of P. falciparum were determined by a Hoechst 33342-based method with a fluorescence-activated cell sorter (FACS) to measure DNA content of infected erythrocytes.¹⁴ Encouragingly, all the compounds displayed submicromolar activities on both Dd2 and 3d7 with broadly similar levels of potencies but with some exceptions (17, 23) (Table 1). In addition, we found excellent differential cytotoxicities in this series, with most compounds associated with a 100-fold or greater selectivity vis-à-vis mammalian Chinese hamster ovary (CHO) cells.

Table 1. Growth Inhibitory Activities of Series A (4–24) on Asexual Ring Stages of P. falciparum Dd2 (Drug Resistant) and 3D7 (Drug Sensitive) and Mammalian Chinese Hamster Ovary (CHO) Cells.

^aNote that R¹/R² are interchangeable due to resonance within the imidazolium ring.

^bConcentration corresponding to 50% of the net amount of fluorescence of nontreated control cultures. Mean (±SD) of n = 3 separate determinations.

^cConcentration required to reduce cell growth by 50% relative to untreated cultures as determined by a tetrazolium dye assay. Mean (±SD) of n = 3 separate determinations.

^dc-C₃H₇ = cyclopropyl.

The most potent compounds were 11, 14, 16, 19 which had low nanomolar activities (IC₅₀ < 10 nM) against both resistant and sensitive plasmodia. Interestingly, the total carbon count on R¹/R² of these compounds was either 5 or 6 out of a possible range of 2–8. Hence, we posited that optimal activity resided within a narrow spectrum, tentatively defined by 5–6 carbons, with declining potencies at either end of this range. Indeed, diminished activity was evident in the dibutyl analog 24 while short hydrophilic fragments like methyl and cyclopropyl were disproportionally represented at R¹/R² of less potent analogs such as 4, 5, 8, and 13. The hydrophilicity of the cyclopropyl fragment, anomalous for its carbon count, is attributed to its unique three-dimensional features.¹⁵

Earlier, we observed significantly diminished antimalarial activity in 3, the non-quinoid analog of YM155 (Figure 1C), which portends a pivotal structure–activity role for the quinone. However, only modest activity was uncovered in representative quinones like 1,4-naphthoquinone, menadione, and 1,4-benzoquinone against NF54 asexual parasites (20–40% inhibition at 1 μM, Table S-2). Hence, we posited that it was not the quinone per se but the fusion of the quinone to the charged imidazolium ring that was required for potent activity. Consequently, we synthesized series B (36–40) in which the quinone is fused to an uncharged alkyl substituted imidazole ring (Scheme 1). Gratifyingly, no member in this series inhibited growth by more than 50% at 1 μM (Table S-2), thus affirming the essentiality of the fused quinone-imidazolium for potent activity.

To further examine the activity moderating effects of the alkyl residues at R¹/R², we prepared series C which has isobutyl at R¹ and longer alkyl side chains (up to dodecyl) at R². These compounds were tested alongside the potent series A analogs (11, 14, 16, 19) on drug sensitive NF4 and drug resistant K1 and W2 strains using the SYBR Green I fluorescence assay as a proxy of parasite proliferation (Table 2).¹⁶ Interestingly, a total carbon count of 5–6 at R¹/R² of series C was yet again associated with optimal activity. Hence, 25 and 26 which have methyl/ethyl at R² have IC₅₀ values that were broadly comparable to 11, 14, 16, 19 of series A. Extending the length of R² caused a gradual and uneven decline in activity.

Table 2. IC₅₀ of Series C (25–35) and Potent Series A Analogs (11, 14, 16, 19) on (i) Asexual Stages of P. falciparum NF54 (Drug Sensitive) and K1, W2 (Drug Resistant) and (ii) Early Gametocytes (EG) and Late Gametocytes (LG) of Transgenic Parasite Line NF54-Pfs16-GFP-Luc.

			IC50 (nM)b			Gametocytocidal IC50 (nM)c
Compound	R1 a	R2 a	NF54	K1	W2	EG	LG
11	C2H5	n-C3H7	3.45 ± 0.31	6.5 ± 1.7	4.94 ± 0.29	390 ± 83	709 ± 112
14	C2H5	n-C4H9	1.64 ± 0.2	2.4 ± 0.7	2.88 ± 0.18	268 ± 56	320 ± 35
16	n-C3H7	i-C3H7	2.28 ± 0.19	3.1 ± 0.8	2.20 ± 0.12	162 ± 52	567 ± 87
19	i-C3H7	i-C3H7	1.43 ± 0.13	2.5 ± 1.3	2.18 ± 0.16	72 ± 5	653 ± 29
25	i-C4H9	CH3	1.92 ± 0.12	3.40 ± 0.40	1.85 ± 0.10	41 ± 17	1088 ± 26
26	i-C4H9	C2H5	1.63 ± 0.02	3.10 ± 0.50	1.62 ± 0.14	33 ± 5	1019 ± 251
27	i-C4H9	n-C3H7	2.97 ± 0.10	4.42 ± 0.10	2.12 ± 0.16	269 ± 48	842 ± 251
28	i-C4H9	i-C3H7	2.36 ± 0.07	4.22 ± 0.21	1.96 ± 0.30	229 ± 79	689 ± 138
29	i-C4H9	c-C3H7	3.94 ± 0.23	5.85 ± 0.08	2.55 ± 0.23	139 ± 48	832 ± 274
30	i-C4H9	n-C4H9	7.20 ± 0.40	9.85 ± 0.21	5.10 ± 0.50	181 ± 87	275 ± 66
31	i-C4H9	i-C4H9	9.19 ± 0.32	12.5 ± 0.7	5.75 ± 0.27	337 ± 65	812 ± 364
32	i-C4H9	n-C6H13	6.20 ± 0.15	10.0 ± 0.1	5.28 ± 0.21	143 ± 9	551 ± 99
33	i-C4H9	n-C8H17	4.36 ± 0.08	8.23 ± 1.02	3.60 ± 0.80	39 ± 18	164 ± 10
34	i-C4H9	n-C10H21	4.96 ± 0.17	13.3 ± 2.1	6.10 ± 0.40	370 ± 73	237 ± 66
35	i-C4H9	n-C12H25	4.30 ± 0.50	11.2 ± 1.0	5.50 ± 0.40	361 ± 75	174 ± 11
Chloroquine			10 ± 3	154 ± 14d	233 ± 40
Artesunate			3.0 ± 0.29	3.26 ± 0.20	2.40 ± 0.20	63 ± 3	259 ± 80
Methylene blue						95 ± 11	143 ± 17

^aR¹ and R² are as depicted in Table 1.

^bConcentration corresponding to 50% of the net amount of SYBR Green I fluorescence of nontreated control cultures. Mean (±SEM) of n = 3 independent biological replicates.

^cConcentration corresponding to 50% reduction of luminescence in early and late gametocytes of NF54-Pfs16-GFP-Luc in a luciferase-based assay. Mean (±SEM) of n = 3 independent biological replicates.

^dIC₅₀ of CQ on the P. falciparum strain (K1) was 212 (±32) on the Hoechst assay, indicating broad equivalence between the Hoechst and SYBR Green I assay platforms.

Next, we assessed the compounds in Table 2 for transmission blocking activity on early (>95% stage II/III) and late (>90% stage IV/V) stage gametocytes.¹⁷ Most compounds were gametocytocidal (IC₅₀ < 1000 nM) and generally more potent against early gametocytes (IC₅₀ = 30–400 nM) than late gametocytes (IC₅₀ = 160–1100 nM). Indeed, several compounds (19, 25, 26, 33) were as cidal as the positive controls artesunate and methylene blue against early gametocytes. There was no discernible trend as to how alkyl chain lengths impacted early gametocytocidal activity in series C. On the other hand, the potent activity of analogs 33 and 35 against late stage gametocytes (comparable to artesunate) alluded to a preference for longer alkyl side chains (total carbon count of ≥12) at R¹/R² against this stage of the life cycle. Noticeably, 33 displayed outstanding potencies against both early and late gametocytes which may suggest its potential in blocking transmission.

The preceding discussion on the effect of R¹/R² alkyl chain length on asexual/sexual potencies pointed to a possible moderating role for lipophilicity. To confirm, the clogP values and antimalarial IC₅₀ values of series A and C were analyzed for statistical correlations based on Spearman ρ coefficients (Tables S-3 and S-4). Interestingly, no significant correlation between clogP and asexual IC₅₀ was detected in series A but it was observed in series C (Table S-4). Very likely, the extended clogP range arising from the inclusion of analogs with longer alkyl side chains in series C led to this outcome. The correlation for series C was positive (smaller clogP/shorter side chains favored potent activity), recapitulating our preceding anecdotal inferences. When correlations between lipophilicities and gametocytocidal activities (IC_50EG/IC_50LG) of series C were examined, none was observed for early gametocytes but a negative relationship was detected for late gametocytes (longer side chains favored potent activity) (Table S-4).

Thus far, most of the compounds in Table 2 qualified as validated antimalarial hits based on their outstanding potencies against asexual (sensitive and multidrug resistant strains, IC₅₀ < 100 nM) and sexual life stages of the parasite.⁵ Progression to early lead status would require evidence of in vivo efficacy (ED₉₀ < 50 mg/kg) against the blood stages of infection in a P. falciparum infected humanized mouse model.⁵ To this end, we selected 19 as a representative member for animal studies. The choice of 19 was primarily motivated by its excellent activity against asexual parasites, exceeding that of the most favored series C analog 33 which is arguably better positioned in terms of gametocytocidal activity.

Prior to pharmacokinetic (PK) profiling in mice, 19 was assessed for its solubility, PAMPA permeability, and liability to metabolic breakdown by rat liver microsomes. 19 had good water solubility (>100 μM, pH 7.4) but no detectable PAMPA permeability. It was remarkably stable to microsomal degradation (Figure S-1) and additionally well tolerated in mice when administered intravenously (2 mg/kg) and orally (20 mg/kg). In line with its hydrophilic nature, the PK of 19 revealed limited tissue distribution (volume of distribution V_d = 8.3 L/kg), rapid clearance from plasma (CL = 70 mL min^–1 kg^–1), and a relatively short half-life (t_1/2 = 1.4 h) (Table 3). Oral bioavailability was however limited at 5%, likely due to its quaternized state which would limit permeability across the gastrointestinal barrier. Notwithstanding, a plot of plasma concentration (after oral administration) versus time showed that levels of 19 were well above its IC_503D7 for the period of exposure (24 h) (Figure 2).

Table 3. PK Parameters for Compound 19 Administered to Male C57BL6 Mice (n = 3 per Dosing Route).

Route	Dose (mg/kg)a	t1/2 (h)	CL (mL min–1 kg–1)	Vd (L/kg)	Cmax (μM)	Tmax (h)	AUC0–last (min·μmol/L)b	F (%)
Intravenous	2	1.4	70	8.3			130
Oral	20	11.3			337	2	69	5

^aMice showed no sign of distress 24 h after single dose administration by either route.

^bAUC was monitored up to 7 h (intravenous) and 24 h (oral).

Figure 2.

Systemic exposure of 19 over 24 h following administration of a single oral dose (20 mg/kg) to healthy mice over 24 h. Dotted lines embedded within plot are the IC₅₀ (1×, 3×) values of 19 against CQ sensitive 3D7 P. falciparum.

Thus encouraged, we proceeded to evaluate the efficacy of 19 on a humanized mouse model of P. falciparum (Pf3D7^0087/N9). Briefly, 19 was administered orally at 5 doses ranging from 1 to 50 mg/kg, with each dose given for 4 days (days 3–6). Parasitemia was determined on each treatment day and on day 7. The effective dose of 19 required to reduce parasitemia by 90% on day 7 (ED₉₀) was estimated to be 34 mg/kg (Figure 3). Noting that parasitemia was not reduced significantly at the highest dose, some caution should be accorded to this value. 19 could admittedly have greater in vivo efficacy if not for its limited oral bioavailability. Appropriate formulation strategies may be employed to circumvent this liability.

Figure 3.

In vivo efficacy of 19 on the humanized P. falciparum (Pf3D7^0067/N9) mouse model. ED₉₀ is the effective dose (mg/kg) required to reduce parasitemia by 90% on day 7 compared to untreated control animals. It was obtained from nonlinear fitting of log parasitemia on day 7 postinfection versus administered oral dose of 19. ED₉₀ was estimated to be 34 mg/kg.

Taken together, we have identified two features in the scaffold that were consequential to potent antimalarial activity. They are (i) substituents (R¹/R²) attached to the ring N atoms and (ii) the fused quinone-imidazolium ring. For (i), alkyl side chains at R¹/R² are preferred to the ring based residues found in YM155, 1 and 2. The lipophilicities imparted by the alkyl chains are important drivers of activity but, as noted earlier, in disparate ways that were influenced by the phase of the plasmodial life cycle. The requirement (ii) for the fused quinone-imidazolium ring is a puzzle. Quinones are found in several antimalarials, and in most instances, they are assumed to disrupt the redox balance in the parasitized erythrocytes through their propensity to redox cycle.¹⁸ Indeed, the potent activity of the antimalarial lead compound plasmadione was attributed to redox cycling by its quinone moiety.¹⁹ In contrast, the low redox potential of the antimalarial drug atovaquone abrogated the role of the quinone in sustaining a subversive substrate-driven redox cycle.²⁰ Rather, atovaquone acts as competitive inhibitor of the plasmodial cytochrome b quinol oxidation (Qo) site,^21,22 with the quinone providing additional sites of contact to the target through hydrogen bonding.²³ This prompted us to evaluate the potent series A analogs (11, 14, 16, 19) against P. falciparum TM90C2B which carries a mutation in the cyt b gene, thus rendering atovaquone inactive against it (Table 4). Interestingly, the compounds were no less active on TM90C2B than on CQ resistant (K1) and sensitive (NF54) strains, thus precluding the mitochondrial cytochrome bc1 complex as a putative target. On the other hand, the same compounds were identified as redox cyclers on the in vitro phenol red horseradish peroxidase assay,²⁴ albeit less effectively than 1,4-naphthoquinone and, intriguingly, exceeding that of the nonquaternized series B compounds which had no detectable redox cycling activity (Table S-5). If these findings are reconfirmed on other assay platforms, it would signal a novel modulating effect by the charged imidazolium on the redox status of the quinone and hence a mechanistic rationale for the structural importance of the quinone-imidazolium entity. Alternatively, noting that YM155 is preferentially taken up by the solute carrier SLC35F2 into malignant cells,²⁵ it is tempting to accord a facilitatory role to the charged quinone-imidazolium entity with regard to uptake into parasitized erythrocytes.

Table 4. IC₅₀ Values of Series A Analogs (11, 14, 16, 19) against P. falciparum NF54, K1, and Atovaquone-Resistant TM90C2B Determined by [³H]-Hypoxanthine Incorporation.

	IC50 (nM)				IC50 (nM)
Compound	NF54 (n = 2)	K1 (n = 2)	TM90C2B (n = 3)	Compound	NF54 (n = 2)	K1 (n = 2)	TM90C2B (n = 3)
11	1.2	2.2	2.2	Chloroquine	8.6	144	140
14	1.0	1.8	1.4	Artesunate	3.2	1.4	2.7
16	1.0	2.1	1.8	Atovaquone	0.4	0.5	11500
19	1.0	1.9	1.6

In conclusion, we have identified the dioxonaphthoimidazolium scaffold as a promising antimalarial pharmacophore, with several members displaying exceptional potencies against sensitive and multidrug resistant asexual parasites (<100 nM) as well as early and late stage gametocytes (<1000 nM). The attractiveness of the scaffold from a medicinal chemistry perspective is further enhanced by its synthetic accessibility, the excellent comparative cytotoxicities of its members, and based on a representative potent analog 19, good aqueous solubility, metabolic stability, and in vivo tolerability in mice. The PK profile of 19 revealed limited tissue distribution, rapid plasma clearance, and a relatively short half-life. However, 19 had limited oral bioavailability (5%) which arguably contributed to its modest activity in the humanized mouse model of P. falciparum. Greater efficacy would conceivably be achieved if not for the poor permeability of 19 arising from its quaternized state. Notwithstanding these PK issues, it is still possible to leverage the inherent merits of the scaffold by engaging formulation strategies or structural modifications to optimize lipophilicity. Lastly, we have provided clarity on the fundamental structure–activity requirements of this class of compounds. Importantly, the quinone is recognized as a necessary but insufficient feature for potent activity. Rather, it is the fused quinone-imidazolium entity that is pivotal, plausibly by facilitating cellular entry or fulfilling a mechanistic role in enhancing the reactivity of the quinone. Additionally, lower lipophilicities appear to augment activity against asexual forms whereas higher lipophilicities favor activity against late stage gametocytes. Future work will focus on addressing the PK limitations of the scaffold through structure modification, establishing a better understanding of the mode of action, and exploring interactions of potent members with other antimalarial agents.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.9b00457.

• Description of methods used to synthesize series A–C compounds; characterization of intermediates and final compounds; purity determination of final compounds; synthesis of target compound 19, NMR and HPLC spectra of 19; activity of series B on asexual stages of NF54 P. falciparum; clogP values of series A and C; Spearman ρ correlation coefficients of series A and C; solubility, PAMPA permeability, and microsomal stability of 19; redox cycling EC₅₀ values of series A and B as determined by the phenol red horseradish peroxidase assay; cytotoxicities of series A compounds on Chinese hamster ovary (CHO) cells; in vitro assessment of antimalarial activity against asexual forms and gametocytes of P. falciparum; pharmacokinetic analysis of 19 in mice; in vivo efficacy of 19 in a humanized mouse model of P. falciparum (PDF)

Author Contributions

^○ S.A., D.C., J.X.T., and K.F. contributed equally. The manuscript was written through contributions of all authors.

The authors declare no competing financial interest.