Dual protonophore-chitinase inhibitors dramatically affect O. volvulus molting.

The L3-stage-specific chitinase OvCHT1 has been implicated in the development of Onchocerca volvulus, the causative agent of onchocerciasis. Closantel, a known anthelmintic drug, was previously discovered as a potent and specific OvCHT1 inhibitor. As closantel is also a known protonophore, we performed a simple scaffold modulation to map out the structural features that are relevant for its individual or dual biochemical roles. Furthermore, we present that either OvCHT1 inhibition or protonophoric activity was capable of affecting O. volvulus L3 molting and that the presence of both activities in a single molecule yielded more potent inhibition of the nematode's developmental process.

Introduction

Onchocerciasis, or river blindness, has plagued over 37 million people worldwide, occurring mostly in the sub-Saharan Africa, Yemen, and isolated areas of Central and South America.[1] The culprit for the infection is the filarial nematode Onchocerca volvulus, which is transmitted to humans through bites of infected blackflies of the genus Simulium. Initial pathological manifestations of the disease include inflammation of the skin and eyes caused by dead or dying microfilariae that eventually results to skin lesions and blindness. Currently, the antiparasitic agent ivermectin is the only drug available for mass treatment.[2] While the drug has proven to be effective in reducing morbidity by killing the microfilariae, it does not target the adult worms and thus does not completely eliminate parasite transmission and infection.[3,4] Additionally, the emergence of ivermectin-resistant O. volvulus(5,6) further necessitates the need to develop therapeutic strategies for the treatment of onchocerchiasis.

Molting is a crucial feature in the life cycle of O. volvulus. The shedding of the L3 cuticle during ecdysis and the proteolytic remodeling of the new L4 cuticle is particularly important for the transition between hosts and infection process of the parasitic nematode. Chitinases act in concert with chitin synthases to facilitate chitin metabolism, a significant process for nematode development.[7] Chitinases have been implicated in remodeling processes during filarial molting and egg hatching.[8,9] In the rodent filarial nematode Acanthocheilonema viteae, RNA interference studies targeting the chitinase gene significantly inhibited the molting of L3 worms.[10] A chitinase from O. volvulus, OvCHT1, was recently identified and found to be solely expressed within the glandular esophagus of the infective L3 larvae and is secreted during postinfective development.[11] OvCHT1 was hypothesized to play roles in molting and parasite transmission and, as such, presents a potential target for pharmacological intervention. From our previous screening efforts, we had disclosed a known veterinary anthelmintic drug, closantel (1), which exhibits potent and selective inhibition toward OvCHT1 (IC50 = 1.60 ± 0.08 μM, Ki = 0.47 ± 0.08 μM).[12] As closantel is a known uncoupler of oxidative phosphorylation,[13] our recent findings implicated a potential bimodal action of the drug. Furthermore, closantel was shown to inhibit the molting of O. volvulus L3 to L4 larvae at 100 μM;[12] however, this profound effect on filarial molting was unclear as to whether it was due to inhibition of OvCHT1 or uncoupling of mitochondrial activity. In the same study, a “retro-fragment”-based approach was conducted to unravel the molecular frameworks that are required for closantel’s chitinase inhibitory activity, in which compound 2 was identified as the relevant structural fragment with a comparable potency as that of closantel (IC50 = 5.8 ± 0.3 μM).[12]

In this study, we constructed a series of closantel analogues to deduce the molecular features that are critical for chitinase inhibition and mitochondrial-uncoupling activity. Because the 3,5-diiodosalicylate moiety is key to closantel’s binding specificity by anchoring it within the OvCHT1 active site,[14] we retained this fragment and focused on scaffold expansion of 2 to generate analogues 3, 4, and 5 (which were easily accessed through amide coupling of carboxylic acids 6 and amine reactants, Figure 1). Simple structural modifications led to the identification of more potent compounds with dual modes of action (as an OvCHT1 inhibitor and a protonophore) as well as analogues acting as chitinase inhibitors only. We also demonstrate that while either OvCHT1 inhibition or mitochondrial uncoupling was effective in abrogating the L3-to-L4 molt, synergistic activities incorporated into a single molecule afforded a more profound impact on molting.

Figure 1

Structures and preparation of closantel analogues.

Results and Discussion

O. volvulus Chitinase Inhibition

The presence of additional (phenolic and amide) protons in closantel may be key for its dual mode of action. For instance, the hydrogen-bonding moieties may be important to affect the required acidic residues of the chitinase catalytic site.[15] To elucidate the relevance of these protons, we initially prepared a simplified version of closantel using a phenoxyphenyl scaffold (compound 3a) and its analogues containing methyl substituents in place of the key hydrogens (compounds 3b–d). As shown in Table 1, compounds 3a, 3b, and 3c have comparable chitinase inhibitory profile as closantel, indicating that the phenolic or amide proton is not necessary for chitinase inhibition. However, simultaneous substitution of both hydrogens with methyl groups (as in the case of compound 3d) resulted to a 2-fold decrease in potency. Exclusion of the hydroxyl moiety (compound 3e) led to a slight increase in inhibitory activity with an IC50 value of 1.06 ± 0.08 μM.

Table 1

IC50 of O. volvulus Chitinase Inhibition

compd	R1	R2	R3	R4	R5	IC50 (μM)
1						1.60 ± 0.08
3a	OH	H	H	H		1.98 ± 0.13
3b	OMe	H	H	H		1.28 ± 0.19
3c	OH	Me	H	H		1.81 ± 0.18
3d	OMe	Me	H	H		3.71 ± 0.14
3e	H	H	H	H		1.06 ± 0.08
3f	OH	H	H	4-Me		1.11 ± 0.03
3g	OH	H	H	4-F		1.57 ± 0.09
3h	OH	H	H	4-Cl		0.70 ± 0.02
3i	OMe	H	H	4-Cl		0.60 ± 0.07
3j	H	H	H	4-Cl		0.87 ± 0.10
3k	OH	H	H	3-Cl		1.74 ± 0.09
3l	OH	H	H	2-Cl		1.20 ± 0.17
3m	OH	H	Cl	4-Cl		0.34 ± 0.04
4a	OH					0.94 ± 0.05
4b	OMe					0.89 ± 0.10
4c	H					1.29 ± 0.03
5a					H	0.47 ± 0.03
5b					Me	0.99 ± 0.16
5c					Cl	0.37 ± 0.03

In an effort to increase the potency against OvCHT1, we constructed a series of derivatives based on compound 3a. From Table 1, it is evident that introduction of the chloro substituent at the para-position of the terminal phenyl ring (compound 3h) enhanced the potency almost 3-fold relative to 3a. The similar IC50 values of compounds 3i, 3j, and 3h further confirmed that neither the phenolic proton nor the hydroxyl functionality is important for chitinase inhibition. Inclusion of a chloro-substituent in the middle phenyl ring (compound 3m, also known as rafoxanide and a known veterinary anthelmintic drug)[16] further improved the potency with an IC50 value of 0.34 ± 0.03 μM and a competitive inhibitory constant (Ki) of 0.13 ± 0.01 μM. We then prepared naphthyl analogues 4a–c and biphenyl compounds 5a–c in an attempt to establish favorable π-stacking and van der Waals interaction with the hydrophobic residues within the chitinase active site to optimize OvCHT1 inhibition. Biphenyl scaffolds have been previously considered as privileged substructures that preferentially interact with diverse proteins.[17] Interestingly, we did observe enhanced potency with the biaryl compounds 4 and 5, with IC50 values for OvCHT1 inhibition as low as 0.37 ± 0.03 μM (for compound 5c). Select compounds that exhibited good potency against OvCHT1 were also evaluated for inhibition of chitinases from other species. Compounds 3h, 3i, 3j, 3m, 5a, and 5c (each at a final concentration of 10 μM) showed complete inhibition of OvCHT1 (see Figure S1 in the Supporting Information) while sparing other chitinases including those from another filarial nematode Brugia malayi (BmCHT1) and protozoans Entamoeba histolytica (EhCHT1) and Plasmodium falciparum (PfCHT1). These results indicate that the analogues are highly OvCHT1-specific and thus are amenable for evaluation in chitinase-specific diseases such as onchocerciasis.

Evaluation of Protonophoric Activity

To examine mitochondrial-uncoupling activity, compounds 1–5 were tested using tetramethylrhodamine ethyl ester (TMRE), a positively charged, mitochondrion-selective dye that serves as a membrane potential sensor. In the presence of a protonophore (e.g., carbonyl cyanide m-chlorophenyl hydrazone or CCCP), the mitochondrion gets depolarized and thus fails to sequester TMRE, resulting in decreased fluorescence intensity. A microplate fluorescence assay was developed to monitor mitochondrial uncoupling of HEK-293T/17 cells in the presence of the inhibitors. Following incubation with the inhibitors at 37 °C, the cells were stained with TMRE and subsequently analyzed by fluorescence spectrometry. As depicted in Figure 2, a dissociable proton (i.e., phenolic hydrogen as in 1, 3a, 3h, and 4a) for the salicylanilide class of compounds is critical for mitochondrial-uncoupling activity. As expected, removal of the hydroxyl moiety (as in compounds 3e, 3j, and 4c) or masking it with a methyl group (compounds 3b, 3i, 3d, and 4b) resulted to a loss of uncoupling activity. We note, however, that the amide proton is equally significant, as substituting it with a methyl group while retaining the hydroxyl moiety (like in the case of compound 3c) eradicated protonophoric activity. Results of mitochondrial-uncoupling activity for the other compounds listed in Table 1 are given in Supporting Information Figure S2. Separate experiments using flow cytometry to analyze membrane polarization were also conducted and the results (see Figure S3 in the Supporting Information) were in agreement to those obtained from the microplate fluorescence assay.

Figure 2

Evaluation of mitochondrial-uncoupling activity using a microplate fluorescence assay. HEK 293T/17 cells were incubated with compound (50 μM) and subsequently stained with TMRE. Data shown as mean fluorescence intensity ± sd (n = 3). Unstained cells (no TMRE) and DMSO were used as negative (−) and positive (+) controls, respectively. RFU = relative fluorescence units (λex = 488 nm, λem = 575 nm).

From the series of derivatives listed in Table 1, we have identified more potent analogues with dual biochemical roles (both as a chitinase inhibitor and a proton ionophore) as well as compounds with chitinase inhibitory activity only. Compounds 3m proved to be the most potent OvCHT1 inhibitor with good mitochondrial-uncoupling activity, while compounds 3i, 3j, and 4b displayed similar chitinase inhibition profile but devoid of protonophoric activity.

Bioaccumulation in the Model Nematode C. elegans

In general, nematodes are resistant to perturbation of small molecules due to the thick cuticles that line their exteriors and oral and rectal cavities.[18,19] The highly insoluble cuticle of O. volvulus is a collagenous framework of proteins that are cross-linked by disulfide bridges.[20] As the bioaccumulation of a drug is correlated to its bioactivity, we deemed it important to investigate the penetrability of the compounds in the nematode. The limited availability of O. volvulus, however, prompted us to consider an alternative model organism, the soil nematode Caenorhabditis elegans. The cuticle of adult C. elegans is composed of multiple layers of collagenous extracellular structures[18] and thus resemble the physical barriers of other nematodes. In fact, due to its easy culturability and rapid life cycle, it was previously used as a model system of bioaccumulation to identify small bioactive molecules.[21] Using a similar approach to address nematode permeability, we initially incubated late-stage L4 worms with compounds 1, 3h, 3i, 3j, 3m, 4a, 4b, 5a, 5c, and CCCP (each at 10 μM final concentration, and equivalent to 2 nmol/mg worm) for 6 h. By LC-MS analysis of worm homogenates, we determined that all the aforementioned compounds accumulated in C. elegans. C. elegans is also equipped with enzymatic xenobiotic defenses that add to its resistance to exogenous pharmacologicals.[22] Similar to what was observed in cattle and sheep,[23] the primary, albeit minor, metabolic pathway of closantel in C. elegans is reductive deiodination, resulting in the formation of two monoiodoclosantel isomers (see Figure S4 in the Supporting Information). Sulfation, glycosylation, and glucuronidation of the salicylate moiety of closantel were not observed in C. elegans homogenates. We add that no effect on the viability of the L4 worms was observed upon incubation with the compounds. We then set out to quantify the concentration of representative compounds in the nematode after 6 and 12 h of incubation. As indicated in Figure 3, compound 3m displayed excellent bioaccumulation in C. elegans, with worm levels of 1.55 ± 0.37 and 1.73 ± 0.11 nmol/mg at 6 and 12 h, respectively, which were >75% of the initial dose of the compound to worms. The concentrations of the naphthyl analogues 4a and 4b were a little less than 50% of the dose at 6 h, and increased to 1.13 ± 0.14 and 1.28 ± 0.08 nmol/mg, respectively, after 12 h. The inability to accumulate at adequate concentrations within the worm can render the compound ineffective no matter how potent it is against the target in vitro. In a survey of over 1000 drug-like small molecules, only less than 10% were shown to penetrate C. elegans at concentrations >50% of the initial doses.[21] Thus, the findings that we observed for the closantel analogues are encouraging because they readily accumulate within the nematode at high levels. We surmise that these compounds also accumulate in the filarial nematode O. volvulus and thus increase the likelihood of interaction with the target protein OvCHT1.

Figure 3

Bioaccumulation of chitinase inhibitors in C. elegans. Late-stage L4 worms were incubated with 10 μM inhibitor (equivalent to 2 nmol/mg worm) for 6 and 12 h. Data shown as mean concentration ± sd (n = 3), expressed in nmol/mg worm.

Inhibition of O. volvulus L3Molting

The target protein OvCHT1 is expressed predominantly in the infective L3 larvae and may be critical in the molting and development of the nematode.[11] Filarial molting of O. volvulus L3 larvae is typically assayed by incubation of the larvae in complete growth medium in the presence of peripheral blood mononuclear cells (PBMCs). To evaluate the efficacy of the inhibitors based solely on their inhibitory properties, the L3 larvae were initially incubated with select compounds (10 μM) for 24 h (to allow sufficient time for accumulation) prior to addition of the growth medium and PBMCs and monitored until day 6 when molting was assessed. Under this assay condition, compounds 3h, 3m, and 4a (all have dual protonophoric and chitinase inhibitory activities) completely abolished molting at 10 μM, as indicated in Figure 4A. Significant effects on molting (up to 84% inhibition) were also observed at a lower concentration of 1 μM for compounds 3h, 3m, and 4a (Figure 4B). Of note, no considerable effect on cell viability was observed when HEK 293T/17 cells were treated with 1 and 10 μM of 3h, 3m, or 4a, for 24 h (see Figure S5 in the Supporting Information). Treatment of L3 larvae with compound 6b (neither an OvCHT1 inhibitor nor a protonophore), showed no inhibition on molting (Figure 4A). To ascertain which activity is relevant for inhibition, we also evaluated compounds 3i and 4b (chitinase inhibitors only) and CCCP (a protonophore only) for their impact on L3 molting. Compounds 3i and 4b have differing effects; while 4b inhibited molting at 10 μM, only marginal inhibition was observed with 3i at the same inhibitor concentration (Figure 4A). The difference in efficacy between the two compounds at 10 μM could not be readily explained as both display similar IC50 values against OvCHT1; however, compound 4b could be accumulating within O. volvulus at higher levels, as was observed in the C. elegans model (vide supra), which might account for its better activity. At a higher concentration of 100 μM, compound 3i completely hindered L3 molting. CCCP also fully inhibited the molting of L3 larvae at 10 μM, suggesting that mitochondrial-uncoupling, by itself, affects this developmental process. Many of the proteins that are necessary for O. volvulus L3 molting are stored within the granules of the esophageal glands.[24] Mitochondrial-uncoupling in the glandular esophagus may, therefore, affect the synthesis of key proteins (including OvCHT1) that are essential for larval development. At a final concentration of 1 μM, both 4b and CCCP did not show any impact on molting (Figure 4B). As both chitinase inhibitory and protonophoric activities are likely required for more potent inhibition, we investigated the effect of incubating L3 larvae with a 1:1 mixture of 4b and CCCP. Whereas 3h, 3m, and 4a were effective at 1 μM, no effect on molting was observed using a combination of 4b + CCCP at 0.5 or 1 μM of each compound (Figure 4C). Treatment with 2.5 μM each of 4b and CCCP was sufficient to completely suppress the L3-to-L4 molt.

Figure 4

Molting of O. volvulus L3 larvae in the presence of inhibitors. Percent molting at (A) 10 μM and (B) 1 μM inhibitor concentration, and (C) in the presence of 1:1 combination of 4b and CCCP each at concentrations shown. Data presented as percent molting in a total of 10 wells containing on average 5–10 larvae per well.

Combination therapy, the simultaneous use of two or more drugs with distinct modes of action and biological targets, has been exploited as a treatment modality in several diseases including HIV,[25] metastatic melanoma,[26] second-stage Trypanosoma brucie gambiense sleeping sickness,[27] and malaria.[28] Despite its efficacy in improving response to medication and minimizing drug resistance, combination therapy is resource-intensive, complicated to administer, and prone to drug–drug interactions and adverse side effects. The utility of “combi-molecules” or single agents with polypharmacological targets has emerged as an alternative strategy, displaying the same advantages while bypassing the drawbacks of drug combinations. The combi-targeting concept is a growing field of interest in the search for therapies against cancer[29,30] and infectious diseases.[31] This dual inhibition approach is well exemplified by compounds 3h, 3m, and 4a, which, through concomitant uncoupling of oxidative phosphorylation and inhibition of OvCHT1, resulted in a more robust inhibition of O. volvulus molting. Moreover, our results demonstrate the superior activity of the dual-targeted single molecules over monotherapy or a dual drug combination (4b + CCCP).

Conclusion

The implications of OvCHT1 in filarial molting has opened an avenue to develop strategies toward the elimination of onchocerciasis. Earlier, DNA immunization with OvCHT1 was shown to provide significant protection against the developing L3 larvae in mice; however, large and frequent doses of DNA were needed to attain the result.[32] The efficacy of the dual mechanistic drug, closantel, has steered us to prepare analogues to further probe the individual effects of OvCHT1 inhibition and protonophoric activity on O. volvulus L3 molting. By subtle structural modifications, we have identified potent bifunctional analogues (3h, 3m, and 4a) as well as OvCHT1 inhibitors only (3i and 4b). These compounds were also found to readily bioaccumulate in the model nematode C. elegans at concentrations up to >75% of the initial dose to worms. Moreover, our results have clearly established the significance of each biochemical role in modulating the O. volvulus L3-to-L4 molt and that synergistic activities (as displayed by 3h, 3m, and 4a) provided a formidable impact on molting. Although these strategies will still need to be validated as potential antifilarial therapies, what we have presented thus far is a notch closer toward the search for effective treatments to combat onchocerciasis.

Experimental Section

Chemistry

General Information. All reactions were performed under nitrogen atmosphere unless otherwise noted. 1H and 13C NMR spectra were recorded on a Bruker DRX-500 or Bruker DRX-600 equipped with a 5 mm DCH cryoprobe. All carboxylic acid and amine starting reactants were obtained from commercial sources except for compounds 6b (used for the syntheses of compounds 3b, 3d, 3i, and 4b), 6c (prepared as described previously[33] and used for syntheses of compounds 3e, 3j, and 4c) and N-methyl-4-phenoxyaniline 7 (used for the syntheses of compounds 3c and 3d).

Compounds 3–5 were prepared according to general procedure A or B.

General Procedure A

A mixture of 3,5-diiodosalicylic acid (1.0 equiv) and thionyl chloride (5 equiv) was heated under reflux for 7 h. After concentration under reduced pressure, cold hexanes were added to precipitate the acid chloride product. The residue was filtered, washed several times with cold hexanes, and air-dried. A solution of the amine reactant (1.0 equiv) and DIPEA (3 equiv) in DMF was added to the acid chloride, and the resulting mixture was stirred at rt for 1 h. The product was subsequently subjected to preparative HPLC purification.

General Procedure B

A solution of 6b or 6c (1.0 equiv), DIPEA (2.0 equiv) and HBTU (1.1 equiv) in DMF was stirred for 15 min at rt. The amine reactant (1.0 equiv) was added, and the resulting mixture was stirred overnight at rt. The product was isolated by preparative HPLC purification. Purity of the final products are generally >95% as assessed by HPLC.

2-Hydroxy-3,5-diiodo-N-(4-phenoxyphenyl)benzamide (3a)

Procedure A, 53% yield. 1H NMR (500 MHz, DMSO-d6) δ 6.99–7.10 (m, 4H), 7.14 (t, J = 7.39 Hz, 1H), 7.36–7.44 (m, 2H), 7.64–7.71 (m, 2H), 8.23 (d, J = 1.90 Hz, 1H), 8.40 (d, J = 1.86 Hz, 1H), 10.66 (s, 1H). 13C NMR (151 MHz, DMSO-d6) δ 81.7, 89.1, 117.3, 118.4, 119.0, 123.4, 123.8, 130.1, 132.8, 135.8, 149.7, 153.5, 156.9, 159.5, 166.7. HRMS-ESI (m/z): [M + H]+ calcd for C19H14I2NO3, 557.9058; found, 557.9058.

3,5-Diiodo-2-methoxy-N-(4-phenoxyphenyl)benzamide (3b)

Procedure B, 60% yield. 1H NMR (600 MHz, CDCl3) δ 3.91 (s, 3H), 6.95–7.05 (m, 4H), 7.10 (t, J = 7.38 Hz, 1H), 7.33 (t, J = 7.94 Hz, 2H), 7.61 (d, J = 8.84 Hz, 2H), 8.24 (d, J = 2.12 Hz, 1H), 8.38 (d, J = 2.13 Hz, 1H), 9.38 (s, 1H). 13C NMR (151 MHz, CDCl3) δ 62.7, 90.1, 93.8, 118.7, 119.8, 121.9, 123.4, 129.4, 129.9, 133.2, 141.3, 150.5, 154.2, 157.1, 157.5, 160.9. HRMS-ESI (m/z): [M + H]+ calcd for C20H16I2NO3, 571.9214; found, 571.9213.

2-Hydroxy-3,5-diiodo-N-methyl-N-(4-phenoxyphenyl)benzamide (3c)

Procedure A, 23% yield. 1H NMR (600 MHz, CDCl3) δ 3.47 (s, 3H), 6.92–7.02 (m, 3H), 7.02–7.09 (m, 4H), 7.13 (t, J = 7.38 Hz, 1H), 7.35 (t, J = 7.91 Hz, 2H), 7.92 (d, J = 1.63 Hz, 1H), 11.89 (s, 1H). 13C NMR (151 MHz, CDCl3) δ 39.7, 79.2, 87.6, 117.7, 119.3, 120.3, 124.0, 128.1, 130.1, 139.2, 139.4, 149.2, 156.8, 157.1, 159.5, 169.2. HRMS-ESI (m/z): [M + H]+ calcd for C20H16I2NO3, 571.9214; found, 571.9205.

3,5-Diiodo-2-methoxy-N-methyl-N-(4-phenoxyphenyl)benzamide (3d)

Procedure B, 58% yield. 1H NMR (600 MHz, CDCl3) δ 3.46 (s, 3H), 3.88 (s, 3H), 6.82 (d, J = 8.78 Hz, 2H), 6.90 (d, J = 7.85 Hz, 2H), 7.01 (d, J = 8.78 Hz, 2H), 7.10 (t, J = 7.40 Hz, 1H), 7.29–7.40 (m, 4H), 7.93 (d, J = 1.98 Hz, 1H). 13C NMR (151 MHz, CDCl3) δ 37.5, 62.7, 87.6, 93.3, 118.9, 119.5, 123.7, 128.3, 130.0, 133.9, 137.4, 138.1, 147.6, 155.9, 156.3, 157.0, 166.7. HRMS-ESI (m/z): [M + H]+ calcd for C21H18I2NO3, 585.9376; found, 585.9366.

3,5-Diiodo-N-(4-phenoxyphenyl)benzamide (3e)

Procedure B, 45% yield. 1H NMR (600 MHz, CDCl3) δ 6.98–7.05 (m, 4H), 7.11 (t, J = 7.40 Hz, 1H), 7.31–7.37 (m, 2H), 7.56 (d, J = 8.73 Hz, 2H), 7.71 (s, 1H), 8.12 (s, 2H), 8.22 (t, J = 1.39 Hz, 1H). 13C NMR (151 MHz, CDCl3) δ 95.2, 118.9, 119.7, 122.3, 123.5, 129.9, 132.7, 135.4, 138.3, 148.4, 154.5, 157.4, 162.7. HRMS-ESI (m/z): [M + H]+ calcd for C19H14I2NO2, 541.9109; found, 541.9106.

2-Hydroxy-3,5-diiodo-N-(4-(p-tolyloxy)phenyl)benzamide (3f)

Procedure A, 51% yield. 1H NMR (600 MHz, CDCl3) δ 2.34 (s, 3H), 6.91–6.95 (m, 2H), 6.98–7.04 (m, 2H), 7.15 (d, J = 8.25 Hz, 2H), 7.44–7.50 (m, 2H), 7.78 (d, J = 1.89 Hz, 1H), 7.87 (s, 1H), 8.18 (d, J = 1.89 Hz, 1H). 13C NMR (151 MHz, CDCl3) δ 20.9, 77.2, 80.3, 89.0, 116.7, 118.9, 119.3, 123.5, 130.5, 130.7, 133.5, 134.3, 151.1, 154.5, 156.0, 160.4, 166.4. HRMS-ESI (m/z): [M + H]+ calcd for C20H16I2NO3, 571.9220; found, 571.9215.

N-(4-(4-Fluorophenoxy)phenyl)-2-hydroxy-3,5-diiodobenzamide (3g)

Procedure A, 43% yield. 1H NMR (600 MHz, CDCl3) δ 6.96–7.08 (m, 6H), 7.49 (d, J = 8.84 Hz, 2H), 7.79 (d, J = 1.44 Hz, 1H), 7.92 (s, 1H), 8.19 (d, J = 1.72 Hz, 1H). 13C NMR (151 MHz, CDCl3) δ 80.3, 89.0, 116.6 (d, J = 23.3 Hz), 116.7, 118.9, 120.8 (d, J = 8.26 Hz), 123.6, 131.1, 134.3, 151.1, 152.7 (d, J = 2.49 Hz), 155.7, 159.1 (d, J = 242.8 Hz), 160.4, 166.5. HRMS-ESI (m/z): [M + H]+ calcd for C19H13FI2NO3, 575.8963; found, 575.8957.

N-(4-(4-Chlorophenoxy)phenyl)-2-hydroxy-3,5-diiodobenzamide (3h)

Procedure A, 46% yield. 1H NMR (600 MHz, DMSO-d6) δ 7.01–7.08 (m, 2H), 7.07–7.14 (m, 2H), 7.41–7.50 (m, 2H), 7.65–7.74 (m, 2H), 8.23 (d, J = 1.80 Hz, 1H), 8.40 (d, J = 1.80 Hz, 1H), 10.69 (s, 1H). 13C NMR (151 MHz, DMSO-d6) δ 81.8, 89.1, 117.4, 119.3, 120.0, 123.8, 127.1, 129.9, 133.2, 135.8, 149.7, 153.0, 155.9, 159.5, 166.7. HRMS-ESI (m/z): [M + H]+ calcd for C19H13ClI2NO3, 591.8668; found, 591.8660.

N-(4-(4-Chlorophenoxy)phenyl)-3,5-diiodo-2-methoxybenzamide (3i)

Procedure B, 23% yield. 1H NMR (600 MHz, CDCl3) δ 3.91 (s, 3H), 6.92–6.96 (m, 2H), 7.01–7.05 (m, 2H), 7.27–7.31 (m, 2H), 7.60–7.66 (m, 2H), 8.26 (d, J = 2.20 Hz, 1H), 8.39 (d, J = 2.20 Hz, 1H), 9.40 (s, 1H). 13C NMR (151 MHz, CDCl3) δ 62.7, 90.1, 93.8, 119.9, 119.9, 122.0, 128.4, 129.3, 129.9, 133.6, 141.3, 150.6, 153.7, 156.2, 157.1, 160.9. HRMS-ESI (m/z): [M + H]+ calcd for C20H15ClI2NO3, 605.8824; found, 605.8817.

N-(4-(4-Chlorophenoxy)phenyl)-3,5-diiodobenzamide (3j)

Procedure A, 44% yield. 1H NMR (600 MHz, CDCl3) δ 6.92–6.97 (m, 2H), 6.99–7.04 (m, 2H), 7.28–7.32 (m, 2H), 7.57 (d, J = 8.78 Hz, 2H), 7.71 (s, 1H), 8.10–8.15 (m, 2H), 8.22 (t, J = 1.35 Hz, 1H). 13C NMR (151 MHz, CDCl3) δ 95.2, 119.8, 120.0, 122.4, 128.5, 129.9, 133.1, 135.4, 138.3, 148.4, 154.0, 156.1, 162.7. HRMS-ESI (m/z): [M – H]− calcd for C17H10I2NO2, 573.8568; found, 573.8556.

N-(4-(3-Chlorophenoxy)phenyl)-2-hydroxy-3,5-diiodobenzamide (3k)

Procedure A, 43% yield. 1H NMR (600 MHz, CDCl3) δ 6.88–6.93 (m, 1H), 7.00 (t, J = 2.12 Hz, 1H), 7.04–7.08 (m, 2H), 7.08–7.12 (m, 1H), 7.23–7.29 (m, 1H), 7.52–7.58 (m, 2H), 7.80 (d, J = 1.83 Hz, 1H), 7.92 (s, 1H), 8.20 (d, J = 1.85 Hz, 1H). 13C NMR (151 MHz, CDCl3) δ 80.4, 89.1, 116.7, 116.9, 119.1, 120.1, 123.5, 123.7, 130.8, 131.9, 134.3, 135.3, 151.2, 154.3, 158.1, 160.4, 166.5. HRMS-ESI (m/z): [M + H]+ calcd for C19H13ClI2NO3, 591.8668; found, 591.8663.

N-(4-(2-Chlorophenoxy)phenyl)-2-hydroxy-3,5-diiodobenzamide (3l)

Procedure A, 35% yield. 1H NMR (600 MHz, CDCl3) δ 6.98–7.02 (m, 2H), 7.03 (dd, J = 1.40, 8.13 Hz, 1H), 7.12 (td, J = 1.44, 7.80 Hz, 1H), 7.23–7.25 (m, 1H), 7.48 (dd, J = 1.54, 8.00 Hz, 1H), 7.49–7.53 (m, 2H), 7.78 (d, J = 1.88 Hz, 1H), 7.87 (s, 1H), 8.19 (d, J = 1.87 Hz, 1H). 13C NMR (151 MHz, CDCl3) δ 80.3, 89.0, 116.7, 118.5, 121.2, 123.5, 125.3, 126.2, 128.2, 131.1, 131.3, 134.3, 151.2, 152.3, 155.0, 160.4, 166.4. HRMS-ESI (m/z): [M + H]+ calcd for C19H13ClI2NO3, 591.8668; found, 591.8662.

2-Hydroxy-3,5-diiodo-N-(naphthalen-2-yl)benzamide (4a)

Procedure A, 27% yield. 1H NMR (600 MHz, DMSO-d6) δ 7.46–7.51 (m, 1H), 7.51–7.55 (m, 1H), 7.78 (dd, J = 2.08, 8.86 Hz, 1H), 7.91 (d, J = 8.58 Hz, 2H), 7.95 (d, J = 8.90 Hz, 1H), 8.25 (d, J = 1.81 Hz, 1H), 8.30 (d, J = 1.61 Hz, 1H), 8.48 (d, J = 1.80 Hz, 1H), 10.84 (s, 1H). 13C NMR (151 MHz, DMSO-d6) δ 81.9, 89.2, 117.6, 118.8, 121.7, 125.5, 126.6, 127.5, 127.6, 128.3, 130.6, 133.1, 134.9, 136.0, 149.7, 159.5, 167.0. HRMS-ESI (m/z): [M – H]− calcd for C17H10I2NO2, 513.8807; found, 513.8800.

3,5-Diiodo-2-methoxy-N-(naphthalen-2-yl)benzamide (4b)

Procedure B, 52% yield. 1H NMR (600 MHz, CDCl3) δ 3.95 (s, 3H), 7.42–7.46 (m, 1H), 7.48–7.52 (m, 1H), 7.57 (dd, J = 2.12, 8.75 Hz, 1H), 7.81 (d, J = 8.11 Hz, 1H), 7.83–7.87 (m, 2H), 8.27 (d, J = 2.19 Hz, 1H), 8.38 (d, J = 1.70 Hz, 1H), 8.44 (d, J = 2.19 Hz, 1H), 9.60 (s, 1H). 13C NMR (151 MHz, CDCl3) δ 62.8, 90.1, 93.9, 117.3, 120.0, 125.5, 126.9, 127.8, 127.9, 129.2, 129.4, 131.1, 134.0, 135.2, 141.4, 150.6, 157.2, 161.1. HRMS-ESI (m/z): [M + H]+ calcd for C18H14I2NO2, 529.9109; found, 529.9114.

3,5-Diiodo-N-(naphthalen-2-yl)benzamide (4c)

Procedure B, 45% yield. 1H NMR (600 MHz, CDCl3) δ 7.44 (ddd, J = 1.09, 6.91, 8.05 Hz, 1H), 7.47–7.51 (m, 1H), 7.57 (dd, J = 2.07, 8.74 Hz, 1H), 7.78–7.86 (m, 3H), 7.89 (s, 1H), 8.17 (d, J = 1.37 Hz, 2H), 8.22 (t, J = 1.46 Hz, 1H), 8.27 (s, 1H). 13C NMR (151 MHz, CDCl3) δ 95.2, 117.6, 120.1, 125.6, 126.9, 127.8, 127.9, 129.2, 131.1, 133.9, 134.8, 135.5, 138.4, 148.4, 162.9. HRMS-ESI (m/z): [M – H]− calcd for C17H10I2NO, 497.8852; found, 497.8847.

N-([1,1′-Biphenyl]-4-yl)-2-hydroxy-3,5-diiodobenzamide (5a)

Procedure A, 72% yield. 1H NMR (600 MHz, DMSO-d6) δ 7.36 (t, J = 7.35 Hz, 1H), 7.47 (t, J = 7.72 Hz, 2H), 7.69 (d, J = 7.31 Hz, 2H), 7.72 (d, J = 8.62 Hz, 2H), 7.78 (d, J = 8.64 Hz, 2H), 8.24 (d, J = 1.75 Hz, 1H), 8.43 (d, J = 1.79 Hz, 1H), 10.73 (s, 1H). 13C NMR (151 MHz, DMSO-d6) δ 81.8, 89.2, 117.5, 122.2, 126.4, 126.9, 127.4, 129.0, 135.9, 136.6, 136.8, 139.5, 149.7, 159.5, 166.9. HRMS-ESI (m/z): [M + H]+ calcd for C19H14I2NO2, 541.9114; found, 541.9116.

2-Hydroxy-3,5-diiodo-N-(4′-methyl-[1,1′-biphenyl]-4-yl)benzamide (5b)

Procedure A, 70% yield. 1H NMR (600 MHz, DMSO-d6) δ 2.34 (s, 3H), 7.27 (d, J = 7.91 Hz, 2H), 7.58 (d, J = 8.03 Hz, 2H), 7.69 (d, J = 8.62 Hz, 2H), 7.76 (d, J = 8.62 Hz, 2H), 8.24 (d, J = 1.65 Hz, 1H), 8.43 (d, J = 1.55 Hz, 1H), 10.70 (s, 1H). 13C NMR (151 MHz, DMSO-d6) δ 20.7, 81.8, 89.2, 117.5, 122.2, 126.3, 126.6, 129.6, 135.9, 136.5, 136.6, 136.6, 136.6, 149.7, 159.5, 166.8. HRMS-ESI (m/z): [M + H]+ calcd for C20H16I2NO2, 555.9270; found, 555.9275.

N-(4′-Chloro-[1,1′-biphenyl]-4-yl)-2-hydroxy-3,5-diiodobenzamide (5c)

Procedure A, 68% yield. 1H NMR (600 MHz, DMSO-d6) δ 7.52 (d, J = 8.46 Hz, 2H), 7.68–7.77 (m, 4H), 7.79 (d, J = 8.61 Hz, 2H), 8.24 (d, J = 1.53 Hz, 1H), 8.42 (d, J = 1.57 Hz, 1H), 10.73 (s, 1H). 13C NMR (151 MHz, DMSO-d6) δ 81.8, 89.2, 117.5, 122.2, 126.9, 128.2, 128.9, 132.2, 135.2, 135.9, 137.1, 138.3, 149.7, 159.4, 166.9. HRMS-ESI (m/z): [M + H]+ calcd for C19H13ClI2NO2, 575.8719; found, 575.8735.

3,5-Diiodo-2-methoxybenzoic Acid (6b)

Methyl iodide (0.80 g, 5.6 mmol) was added dropwise to a mixture of 3,5-diiodosalicylic acid (1.0 g, 5.1 mmol) and K2CO3 (0.90 g, 6.5 mmol) in acetone (10 mL). After stirring for 3 h at rt, the solvent was removed in vacuo, and the residue was partitioned between H2O and CH2Cl2. The organic layer was separated, and the aqueous phase was extracted with CH2Cl2. The organic extracts were concentrated under reduced pressure to give crude methyl 3,5-diiodo-2-methoxybenzoate. The crude product was dissolved in hot methanol (10 mL) to which 1 M KOH (10 mL, 10 mmol) was added. The resulting mixture was heated under reflux for 2 h, diluted with water, and neutralized by addition of 1 M HCl. The product was collected by filtration and recrystallized with ethanol to give 6b in 61% yield (0.62 g) over two steps. 1H NMR (600 MHz, DMSO-d6) δ 3.76 (s, 3H), 7.95 (d, J = 2.09 Hz, 1H), 8.30 (d, J = 2.08 Hz, 1H). 13C NMR (151 MHz, DMSO-d6) δ 61.9, 89.2, 96.6, 128.7, 139.2, 149.2, 158.1, 165.1. HRMS-ESI (m/z): [M – H]− calcd for C8H5I2O3, 402.8328; found, 402.8325.

N-Methyl-4-phenoxyaniline (7)

Compound 7 was synthesized according to the literature procedure.[34] To a cooled solution of N-(4-phenoxyphenyl)acetamide (0.50 g, 2.2 mmol) in anhydrous THF (5 mL) was added NaH (97 mg, 2.4 mmol, 60% dispersion in mineral oil). After stirring for 5 min, MeI (0.20 mL, 3.2 mmol) was added dropwise, and the resulting mixture was stirred for 1 h at rt and then partitioned between H2O and CH2Cl2. The organic phase was separated and the aqueous layer was extracted with CH2Cl2. Concentration of the combined organic extracts in vacuo gave the crude N-methyl-N-(4-phenoxyphenyl)acetamide, which was used in the next step without further purification. Concentrated HCl (0.5 mL) was added to a stirred solution of crude N-methyl-N-(4-phenoxyphenyl)acetamide in ethylene glycol (3 mL). The mixture was heated under reflux overnight, diluted with H2O, and extracted with EtOAc. The organic layers were combined, dried over anhydrous Na2SO4, and concentrated under reduced pressure. Purification of the crude product by silica chromatography gave compound 7 in 56% yield (0.25 g) over two steps. 1H NMR (600 MHz, CDCl3) δ 2.85 (s, 3H), 6.61–6.65 (m, 2H), 6.94–6.98 (m, 4H), 7.03 (t, J = 7.35 Hz, 1H), 7.30 (t, J = 7.96 Hz, 2H). 13C NMR (151 MHz, CDCl3) δ 31.4, 113.5, 117.1, 121.4, 122.0, 129.6, 146.1, 147.6, 159.3. HRMS-ESI (m/z): [M + H]+ calcd for C13H14NO, 200.1075; found, 200.1069.

N-(2-(1H-Indol-3-yl)ethyl)-2-hydroxy-3,5-diiodobenzamide (8)

Procedure A, 65% yield. 1H NMR (500 MHz, DMSO-d6) δ 2.97 (t, J = 7.45 Hz, 2H), 3.57 (q, J = 7.04 Hz, 2H), 6.94–7.02 (m, 1H), 7.03–7.11 (m, 1H), 7.18 (d, J = 2.21 Hz, 1H), 7.34 (d, J = 8.09 Hz, 1H), 7.55 (d, J = 7.86 Hz, 1H), 8.14–8.23 (m, 2H), 9.28 (t, J = 5.43 Hz, 1H), 10.84 (s, 1H). 13C NMR (151 MHz, DMSO-d6) δ 24.6, 40.3, 81.4, 88.8, 111.4, 111.5, 116.3, 118.2, 118.3, 121.0, 122.8, 127.1, 135.2, 136.2, 149.3, 159.9, 168.0. HRMS-ESI (m/z): [M + H]+ calcd for C17H14I2N2O2, 532.9218; found, 532.9208.

Chitinase Inhibition

Chitinase activity assay was performed as previously described.[12]

Mitochondrial-Uncoupling Assays

Adherent human epithelial kidney cells [HEK 293T/17] (ATCC CRL-11268) were trypsinized and resuspended in 10% FBS in DMEM at a density of 1–2 × 106 cells/mL. Cells were incubated with DMSO (blank control) or 50 μM compound for 30 min at 37 °C. Tetramethylrhodamine ethyl ester perchlorate (TMRE, ∼450 nM final concentration) was added, followed by incubation in the dark for 30 min at 37 °C. Cells were washed twice with 0.2% BSA in PBS, centrifuged, and resuspended in 0.2% BSA in PBS. For the microplate assay, fluorescence signals were measured using a SpectraMaxM2e microplate reader (Molecular Devices, Sunnyvale, CA) (λex = 549 nm, λem = 575 nm). For flow cytometry, 1–2 × 106 cells per sample were collected using DIVA 6.0 software on an LSRII flow cytometer (BD Biosciences, San Jose, CA). FACS data was further analyzed using FlowJo software (Tree Star, Inc., Ashland, OR).

C. elegans Culture

C. elegans growth and maintenance were performed using standard protocols.[35]

Bioaccumulation in C. elegans

The procedure for the small molecule accumulation assay was adapted from Burns et al.[21] Briefly, synchronized worms were grown to late-stage L4 worms at 25 °C on Escherichia coli OP50 for 40 h. Worms were harvested, washed three times with water, and resuspended in M9 buffer to a final concentration of ∼5–10 worms/μL. To 200 μL of the worm suspension was added 2 μL of 1 mM compound in DMSO (equivalent to ∼2 nmol/mg worm). After incubation for 6 h (or 12 h) at rt, the worms were washed with 500 μL of 0.1% SDS, followed by three 500 μL washes with water. After washing, the worms were flash frozen in liquid nitrogen, lyophilized, weighed, and stored at −80 °C until analyses.

Sample Analysis

Worms were homogenized for 5 min in a 100-volume equivalent of cold methanol/acetonitrile mixture (1:1) containing 500 nM internal standard 8, using a bullet blender (Next Advance, Inc., Averill Park, NY). After centrifugation for 10 min at 4 °C, the supernatants were collected and analyzed by reversed-phase LC with MS detection (mass filter at m/z = 589.846–589.856 for 3h, m/z = 605.878–605.888 for 3i, m/z = 623.807–623.817 for 3m, m/z = 513.875–513.885 for 4a, and m/z = 529.906–529.916 for 4b). Standard calibration curves were prepared by fortification of blank worms with compounds 3h, 3i, 3m, 4a, and 4b at concentrations up to 2 nmol/mg worm. The concentrations in worms were quantified using peak area ratios relative to the internal standard and linear regression parameters were calculated from the calibration curve standards prepared in blank worms.

Liquid Chromatography and Mass Spectrometry

The LC system consisted of Agilent 1200 LC (Agilent Technologies, Santa Clara, CA) equipped with an autosampler and a column heater. Mass spectrometric experiments were performed with Agilent TOF 6210 and monitored with Mass Hunter qualitative analysis software, version B.03.01 (Agilent Technologies, Santa Clara, CA). Mass spectrometry acquisition was performed using electrospray ionization with the following parameters: capillary voltage = 3.5 kV, nebulizer pressure = 20 psig, drying gas flow = 7 L/min, and gas temperature = 350 °C. Samples were analyzed on a Poroshell 120 EC-C8 column (2.7 μm, 2.1 mm i.d. 100 mm, Agilent, USA). The mobile phase consisted of elution at 0.20 mL/min starting with an 8 min linear gradient from 70% A/30% B to 100% B and then 100% B for 12 min (positive-ESI, A = 0.1% formic acid in water, B = 0.1% formic acid in acetonitrile; negative-ESI, A = water, B = acetonitrile).

O. volvulus L3 Molting Inhibition

Experiments using O. volvulus L3 larvae in molting assay were the same as described previously[12] except that the larvae were incubated with the compounds for 24 h prior to addition of the complete medium (containing 20% heat inactivated FCS) and normal PBMCs.

Cell Viability Assay

HEK 293T/17 cells (2 × 104 cells) were plated in 96-well plates and incubated at 37 °C for 24 h. Cells were then treated with compounds 3h, 3m, and 4a (at a final concentration of 1 and 10 μM), and an MTS assay was performed at 0.5 and 24 h postincubation using the CellTiter 96 aqueous non-radioactive cell proliferation assay (Promega, Madison, WI) per manufacturer’s instructions.