Antiparasitic lethality of sulfonamidebenzamides in kinetoplastids

Abstract

A sulfonamidebenzamide series was assessed for anti-kinetoplastid parasite activity based on structural similarity to the antiparasitic drug, nifurtimox. Through structure-activity optimization, derivatives with limited mammalian cell toxicity and increased potency toward African trypanosomes and Leishmania promastigotes were developed. Compound 22 had the best potency against the trypanosome (EC₅₀ = 0.010 μM) while several compounds showed ~ 10-fold less potency against Leishmania promastigotes without impacting mammalian cells (EC₅₀ > 25 μM). While the chemotype originated from an unrelated optimization program aimed at selectively activating an apoptotic pathway in mammalian cancer cells, our preliminary results suggest that a distinct mechanism of action from that observed in mammalian cells is responsible for the promising activity observed in parasites.

Graphical abstract

The Trypanosomatida order of flagellated protists, including the African trypanosome Trypanosoma brucei and several Leishmania spp., are responsible for widespread human diseases that include African sleeping sickness¹ and leishmaniasis,² respectively. Together, these diseases infect over 2 million people and place a tremendous burden on the local medical infrastructure where they occur. Therapeutic options for these parasitic diseases are limited by toxicity and efficacy, with the rising specter of emerging resistance increasing the possibility that the suboptimal therapeutics currently in use will soon be obsolete. Consequently, finding novel, safe and effective antiparasitic agents is critical to bridge the chasm between available options and none at all. The recent disclosure of GNF6702, a selective inhibitor of the kinetoplastid proteasome that imparts broad spectrum activity,³ shows progress on this front. For our own efforts, we investigated the possibility of targeting multiple parasites to reveal a distinct, sulfonamide-based compound series that potently impacts both T. brucei and Leishmania trypanosomatid parasites.

Our sulfonamidebenzamide-based compound library originated from an orthogonal medicinal chemistry program aimed at detecting agents that selectively upregulated the expression of the C/EBP-homologous protein (CHOP) in cancerous mammalian cells.⁴ Increased expression of CHOP, which occurs in normal cells in response to unresolved, misfolded protein accumulation, ultimately leads to programmed cell death (PCD) in mammals if cellular homeostasis is not otherwise achieved.^5–6 While there is evidence that singled-celled protists can respond to stimuli and initiate a programmed cellular response similar to metazoan apoptosis, the noted absence of clear homologs to the caspases involved suggest alternative mechanisms may be engaged or that the phenotypes that are similar to the PCD-based program are due to “incidental death”.^7–12 Our interest in these compounds as potential antiparasitic agents was piqued by the realization that the series, represented by compound 1, shared some common skeletal features with anti-trypanosomal drug, nifurtimox (Lampit®, 2, Fig. 1). Both compounds bear a nitrofuryl group that is conjugated to an adjacent pi-system (blue shaded region) which is tethered to a sulfone or sulfonamide (green shaded space) through a nitrogen-carbon framework (beige shaded area).

Figure 1.

Similarity between scaffold 1 and nifurtimox 2

The exact mechanism of action of nifurtimox has not been elucidated, though studies propose at least two alternative pathways involving the induction of oxidative stress leading to apoptosis^13–16 or reductive activation by a eukaryotic type I nitroreductase^17–19 – both of which implicate the involvement of the nitrofuryl moiety as a warhead. Given the structural congruency between these templates and the ambiguity of mechanisms involved with each, we decided to investigate if these compounds harbored antiparasitic activity.

Sulfonamidebenzamides were synthesized as previously described.⁴ Structural integrity was confirmed by ¹H and ¹³C NMR, and purity was validated to be > 95% by UV/LCMS. Compounds were assessed for their impact on parasite viability in 96- or 384-well plate format against T. brucei bloodstream form (BSF)^20–22 and L. amazonensis promastigote form parasites,²³ respectively (Table 1). Immortalized cell lines which are commonly used to assess general mammalian cytotoxicity are hypersensitive to apoptotic pathway modulators.^4,24 As many of these compounds were originally developed as inducers of the mammalian cell apoptotic CHOP pathway, immortalized cell lines were not useful indicators of general cytotoxicity. As a result, we utilized a non-immortalized, human foetal lung myofibroblast cell line, IMR90, to detect cytotoxic effects on non-parasite cells.²⁵ All compounds were determined to have EC₅₀ values > 25 μM on IMR90 cells.

Table 1.

Sulfonamidebenzamide structure-activity data against T. brucei and L. amazonensis¹,²

compound	central ring substitution	R1	R2	cytotoxicity EC50 (μM)3	T. brucei EC50 (μM)4	T. brucei selectivity index, SI5	L. amazonensis EC50 (μM)6	L. amazonensis selectivity index, SI5
1	1,4	N-morpholine	5-NO2-furyl	> 25	0.46 ± 0.05	> 54	3.0 ± 0.105	> 8
2	1,4	N-piperidine	5-NO2-furyl	> 25	0.22 ± 0.06	>114	13.7 ± 7.0	> 2
3	1,4	4-CH3-N-piperidine	5-NO2-furyl	> 25	0.12 ± 0.02	> 208	7.7 ± 5.0	> 3
4	1,4	4,4-dimethyl-N-piperidine	5-NO2-furyl	> 25	0.09 ± 0.01	> 278	0.28 ± 0.1	> 89
5	1,4	4-t-butyl piperidine	5-NO2-furyl	> 25	0.03 ± 0.02	> 833	1.4 ± 0.3	> 18
6	1,4	N-3-azaspiro[5.5]undecane	5-NO2-furyl	> 25	0.03 ± 0.00	> 833	1.5 ± 0.9	> 17
7	1,4	4-OH-N-piperidine	5-NO2-furyl	> 25	0.31 ± 0.07	> 81	4.4 ± 1.8	> 6
8	1,4	4-Cl-N-piperidine	5-NO2-furyl	> 25	0.08 ± 0.04	> 313	7.9 ± 2.8	> 3
9	1,4	4-F-N-piperidine	5-NO2-furyl	> 25	0.42 ± 0.03	> 60	7.4 ± 2.5	> 3
10	1,4	4-NH-piperazine	5-NO2-furyl	> 25	0.73 ± 0.03	> 34	10.5 ± 4.3	> 2
11	1,4	cyclohexyl	5-NO2-furyl	> 25	0.49 ± 0.05	> 51	1.6 ± 0.7	> 16
12	1,4	4-pyran	5-NO2-furyl	> 25	0.53 ± 0.04	> 47	0.3 ± 0.05	> 83
13	1,4	phenyl	5-NO2-furyl	> 25	0.11 ± 0.02	> 227	4.3 ± 1.8	> 6
14	1,4	N-morpholine	2-furyl	> 25	> 10	NA	> 25	NA
15	1,4	N-morpholine	2-thiophene	> 25	> 10	NA	> 25	NA
16	1,4	N-morpholine	phenyl	> 25	> 10	NA	> 25	NA
17	1,4	N-morpholine	5-CH3-furyl	> 25	> 10	NA	> 25	NA
18	1,4	N-morpholine	4-NO2-phenyl	> 25	> 10	NA	> 25	NA
19	1,4	4,4-dimethyl-N-piperidine	3-NO2-phenyl	> 25	6.6 ± 0.15	> 4	13.3 ± 6.2	> 2
20	1,4	4,4-dimethyl-N-piperidine	5-CF3-furyl	> 25	7.55 ± 1.46	> 3	10.7 ± 1.0	> 2
21	1,4	N-morpholine	5-NO2-thiophene	> 25	0.060 ± 0.00	> 417	1.3 ± 0.03	> 19
22	1,4	4,4-dimethyl-N-piperidine	5-NO2-thiophene	> 25	0.010 ± 0.001	> 2500	0.1 ± 0.01	> 250
23	1,4	4-t-butyl piperidine	5-NO2-thiophene	> 25	0.030 ± 0.001	> 833	0.1 ± 0.01	> 250
24	1,4	N-3-azaspiro[5.5]undecane	5-NO2-thiophene	> 25	0.020 ± 0.000	> 1250	0.1 ± 0.01	> 250
25	1,3	4,4-dimethyl-N-piperidine	5-NO2-thiophene	> 25	0.045 ± 0.001	> 556	1.3 ± 0.1	> 19
26	1,3	4,4-dimethyl-N-piperidine	5-NO2-furyl	> 25	0.32 ± 0.001	> 78	0.1 ± 0.02	> 250
27	1,3	4,4-dimethyl-N-piperidine	2-furyl	> 25	5.11 ± 0.86	> 5	1.2 ± 0.1	> 21
28	1,3	4,4-dimethyl-N-piperidine	3-NO2-phenyl	> 25	10.39 ± 1.06	> 2	3.0 ± 0.03	> 8
29	1,3	N-morpholine	5-NO2-furyl	> 25	1.47 ± 0.19	> 17	0.9 ± 0.1	> 28

¹Experimental data is an average of at least 3 runs (n ≥ 3);

²Nifurtimox as a control afforded T. brucei EC₅₀ = 7.5 ± 0.05 μM, L. amazonensis EC₅₀ = 0.8 ± 0.1 μM, IMR90 EC₅₀ > 25 μM;

³Mammalian cytotoxicity was determined using IMR90 cells;

⁴Bloodstream form parasites;

⁵SI = IMR90 EC₅₀/parasite EC₅₀;

⁶Promastigote stage parasites.

NA = not applicable.

Generally, T. brucei BSF parasites were more susceptible to this class of compounds than L. amazonensis promastigotes with distinguishable structure-activity relationships (SAR) apparent for each parasite. Analogs bearing structural variants in the 4-position of the N-morpholine sulfonamide of 1 showed potent cidal activity on T. brucei BSF parasites with EC₅₀ values ranging from 0.03 – 0.73 μM (compounds 1–10, Table 1). A broader and comparatively weaker activity profile was observed for the same compounds when evaluated against L. amazonensis, though compound 4 was highlighted as one of the more potent members with EC₅₀ = 0.28 μM. Sulfone derivatives 11–13 were also effective in the submicromolar range against T. brucei, and sulfone 12 demonstrated nearly equal potency against both parasites. Analogs 14–18 were surveyed with a modified nitrofuryl moiety. Augmentation, in all cases, resulted in loss of potency against both parasites. Notably, compounds 19 and 20 showed no mammalian CHOP activity,⁴ though moderate micromolar potency against parasites was observed with a 3-nitrophenyl group of 19 and interestingly, with the non-nitro group containing 5-trifluoromethylated acylfuran unit in 20. Acylnitrothiophenes 21–25 were very potent against T. brucei, with little potency fluctuation observed for structural changes in the N-morpholine sulfonamide portion or when the 1,4-substitution pattern of the central phenyl ring was exchanged for a 1,3-arrangement, as shown in 25. Analogously for these derivatives, the previously observed preference for a nonpolar alkyl group in the 4-position of the sulfonamide moiety was underscored against T. brucei. The best anti-Leishmania activity was also observed in this group with several compounds showing potencies of 100 nM. Ultimately, the effort revealed that, for T. brucei, the 1,4-substitution of the phenyl ring was preferred in combination with a 5-nitrofuryl- or 5-nitrothiophenyl amide group and a cyclic sulfonamide bearing a lipophilic 4-positioned substituent. Against T. brucei parasites, many analogs were generated that demonstrated low nanomolar potency. For L. amazonensis promastigotes, acylnitrothiophenes were generally the most potent representatives with an analogous preference for alkyl substitution on the distal end of the sulfonamide moiety.

Given that the activity profile against each parasite could be dramatically improved in a tractable manner, we turned our attention to finding evidence of a similar or disparate mechanism of action between the parasites and mammalian cells. Previous researchers have described a PCD-like response in kinetoplastid parasites, with stimuli such as the lectin concanavalin A,^26–27 prostaglandin D²⁸, or unresolved ER stress initiating phenotypes consistent with PCD, including alteration in cellular DNA content due to disruption of replication, DNA degradation, and surface membrane blebbing^{12, 29}.

To explore the mode of action of subject compounds, parasites were scored for loss of membrane phospholipid asymmetry, a hallmark of early apoptosis in mammalian cells. Phosphatidylserine (PS) translocation was detected using an Annexin V-FITC Apoptosis Detection Kit (Enzo Life Sciences, Inc.) Parasites were incubated with test compounds for 6–24 hr (with time points established to limit necrotic/dying cells to less than 0.5% of the population), equilibrated with manufacturer-provided binding buffer, and labeled with Annexin V-FITC (at a 1:200 dilution of the provided stock) along with propidium iodide (PI, 2 μg/mL) to detect necrosis and eliminate dead cells from consideration. Using flow cytometry to analyze parasite staining, 10,000 BSF trypanosomes (a number sufficient to establish statistical significance in observed differences) were scored for changes in Annexin-V labeling after exposure to compound 5 for 6 h at its EC₅₀ value. Compound-treated parasites showed an increase in Annexin-V labeling after exposure (Fig. 2), with a relative fluorescence median signal of 1225 compared to that of the control line, 463. A second structurally related, though less potent, trypanotoxic mammalian apoptotic activator, compound 7, yielded similar changes in Annexin-V labeling (data not shown). Procyclic form (PF) parasites were similarly responsive, with Annexin-V treatment yielding a mean relative fluorescence of 658 and 849 for the untreated and treated lines, respectively (data not shown). These experiments show that treated parasites demonstrate similar cell membrane rearrangements as observed during early apoptosis events in mammalian cells, though the studies do not give an indication of what pathway may be engaged. Deconvoluting the specific pathway by which the phenotype is induced will require a substantially more in depth effort.

Figure 2.

Analysis of T. brucei BSF 90-13 parasites labeled by Annexin-V after treatment with compound 5. The mean peak fluorescence of the control population is indicated (vertical line).

The activators of the mammalian apoptotic branch of the UPR were found to be potent anti-parasitic compounds with selectivity for parasites sufficiently high to suggest that these compounds could prove useful in future therapeutic development. Because the agents trigger apoptotic-like responses in the African trypanosome, it is tempting to speculate that the mode of action is similar in both mammals and trypanosomes. However, the structure-activity assessment identified compounds that did not retain activity against the mammalian CHOP pathway, thereby failing to induce mammalian apoptosis – though the compounds retained activity in killing parasites. This result, combined with the possibility of “incidental death”,¹⁰ suggests that the small molecules yielded PCD-like behavior as a consequence of targeting pathways that yield similar lethal phenotypes, an issue we look forward to resolving in the future as a more robust SAR effort and mechanistic study is undertaken.