Article pubs.acs.org/Organometallics
Synthesis, Characterization, and Biological Activity of Ferrocenyl Analogues of the Anthelmintic Drug Monepantel Jeannine Hess,† Malay Patra,† Vanessa Pierroz,†,‡ Bernhard Spingler,† Abdul Jabbar,§ Stefano Ferrari,‡ Robin B. Gasser,*,§ and Gilles Gasser*,† †
Department of Chemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland Institute of Molecular Cancer Research, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland § Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, Victoria 3010, Australia ‡
S Supporting Information *
ABSTRACT: There is major demand for the development of structurally new anti-infectives using innovative approaches to circumvent multidrug resistance in parasites. Herein, we describe the synthesis and characterization of ferrocenyl precursors and derivatives (2−8) of an anthelmintic drug, monepantel. All compounds were isolated as their racemates and characterized by 1H, 13C, and 19F NMR spectroscopy, mass spectrometry, and IR spectroscopy. The purity of individual compounds was confirmed by elemental microanalysis. The molecular structures of three of the organometallic compounds (5−7) were also established by X-ray crystallography. The biological activities of these compounds were then evaluated in vitro on various important eukaryotic parasites, including H. contortus, T. colubriformis, and D. immitis. The potencies against D. immitis (canine heartworm) of two compounds, a ferrocenecontaining precursor (4) and the final ferrocene-based monepantel derivative (8), were shown to be moderate (EC50 = 3.70 μg/ mL for 4 and 5.60 μg/mL for 8) and were comparable with those of the controls AAD85 (EC50 = 2.20 μg/mL) and a commercial drug, ivermectin (EC50 = 1.00−3.00 μg/mL). The assessment of the cytotoxicity using cancerous HeLa and noncancerous MRC5 cell lines revealed that these compounds have moderate to low toxicities in mammalian cells, thereby showing selective activity on parasites.
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targeting nematodes, since nAChr is absent from mammals.8−10 Although monepantel or, in general, AADs represent a class of anthelmintics with a “new” mode of action, nematodes with a reduced sensitivity to monepantel are already emerging,11−13 as postulated earlier using an in vitro selection procedure14 and later confirmed in field studies.11−13 Considering the current treatment and control options for parasitic worms, there is an urgent need for the development of novel and structurally different classes of potential anthelmintics.4 With this in mind, our research groups have recently initiated a program to modify monepantel with various organometallic moieties.15 This strategy was already proven to be successful in various areas of medicinal research16−24 and for the design of anticancer,25−27 antimalaria,21,28 antitrypanosome,29−39 antibacterial,40 antifungal,41 and antischistosome drugs, to name a few.42−46 Two prominent examples where organometallic derivatization has improved and modulated the activity profile of the original drug are ferroquine and ferrocifen, the ferrocenyl analogues of the antimalaria drug chloroquine and anticancer drug tamoxifen, respectively.24,47 In both cases, the incorporation of the ferrocene fragment has brought an additional metal-specific mode of action. In general, the
INTRODUCTION Over the last decades, controlling parasitic diseases of animals has become a growing concern and a challenge worldwide. On one hand, diseases of livestock animals caused by gastrointestinal roundworms (nematodes), such as Haemonchus and Trichostrongylus species, are responsible for substantial production losses due to morbidity and mortality.1,2 On the other hand, diseases of small animals (e.g., dogs and cats) caused by parasites, such as D. immitis (canine heartworm), are highly significant clinically and cause considerable suffering.3 Thus, the availability of effective curative and preventative treatments is central to the control and management of parasitic diseases. To date, control has relied on the use of anthelmintic drugs of four main classes (benzimidazoles, imidazothiazole, macrocyclic lactones, and amino-acetonitrile derivatives). However, the overusage of some of these drugs has led to the emergence of drug and sometimes multidrug resistance problems in parasites, compromising control in many countries.4 A promising anthelmintic, namely monepantel, which belongs to the class of amino-acetonitrile derivatives (AADs; see Scheme 1), was recently commercialized for the treatment of gastrointestinal nematodes in livestock.5−7 Monepantel selectively targets the nematode-specific nicotinic acetylcholine receptor (nAChr) subunit, which is of major interest for © XXXX American Chemical Society
Received: July 19, 2016
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DOI: 10.1021/acs.organomet.6b00577 Organometallics XXXX, XXX, XXX−XXX
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Organometallics
Scheme 1. Structure of Monepantel (AAD 1566) and Design of Organometallic Derivatives of Monepantel Using Three Different Strategiesa
a
OM = ferrocene, ruthenocene or cymantrene; FG = SCF3, F, Cl, Br, I, SCH3, CF3, OCF3, S(O)CF3, S(O)2CF3.
Scheme 2a
a Reagents and conditions: (i) t-BuLi, DMF, Et2O, 35 min, room temperature, 98%; (ii) acetic anhydride, reflux, 2 h, 74%; (iii) hydroxylamine chlorhydrate, dry EtOH, NaOH, reflux, 3 h, 78%; (iv) LiAlH4, dry THF, overnight, room temperature, 51%; (v) NEt3, 4-(trifluoromethylthio)benzoyl chloride, dry THF, overnight, room temperature, 40% (6, dilute conditions) and 7% (7), respectively; (vi) NaH, 3-fluoro-4(trifluoromethyl)benzonitrile, dry THF, overnight, 0 °C → room temperature, 79%.
organometallic derivatives of monepantel, which we designed using two different strategies (Scheme 1, strategy I/II).15 In that initial study, we substituted the aryloxy part with different organometallic moieties (ferrocene, ruthenocene, and cymantrene) and varied the functional groups at the benzamide unit (SCF3, F, Cl, Br, I, SCH3, CF3, OCF3, S(O)CF3, and S(O)2CF3) of monepantel; 9 of the 27 compounds synthesized using strategy I/II showed moderate activities against H. contortus and T. colubriformis. Interestingly, we also found that a few of our organic/organometallic derivatives exhibited strong efficacy against microfilariae of D. immitis, which causes heartworm disease in companion animals, particularly dogs. In contrast to our previous design, in this study we envisaged to replace the chiral C2 spacer (point chirality) by a chiral
favorable redox properties of the Fe(II) core of ferrocene potentiates the generation of toxic reactive oxygen species (ROS) through a Fenton type of reaction. Additionally, the attachment of ferrocene enhances the lipophilicity of organic drugs, thereby modulating the ADMET (adsorption, distribution, metabolism, excretion, and toxicity) properties of the original organic compounds. For example, ferroquine is active against chloroquine-resistant Plasmodium falciparum (P. falciparum) strains where chloroquine is inactive.21,28,48 The organometallic derivatization of tamoxifen expanded the activity profile of the original drug and rendered it active against both estrogen-positive and -negative breast cancers.47,49 Recently, we have reported the synthesis, characterization, and biological evaluation of a series of organic precursors and B
DOI: 10.1021/acs.organomet.6b00577 Organometallics XXXX, XXX, XXX−XXX
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Organometallics Scheme 3. Proposed Acid-Catalyzed Reaction Mechanism for the Formation of 7 from 6
aldoxime functional group. Furthermore, a peak at m/z 282 in the ESI-MS spectrum (positive detection mode) corresponding to [4 + Na]+ was observed. The aldoxime group of 4 was then reduced to the corresponding amine using lithium aluminum hydride, and 5 was obtained in 51% yield. With the bifunctionalized ferrocenyl intermediate 5 in hand, we focused on the amide bond formation with commercially available 4(trifluoromethylthio)benzoyl chloride under alkaline conditions to obtain compound 6. In our initial attempt, we obtained N(2-hydroxymethyl)ferrocenyl)-4-((trifluoromethyl)thio)benzamide (6) as a minor product with yields 60% at a concentration of 32 μg/mL (32 ppm) to qualify for further testing. Activity in Vitro against C. felis. Oral Test. This test was conducted as described by Wade et al. and Zakson-Aiken et al.64,65 In brief, adult fleas were placed in a suitably formatted microtitration plate, allowing fleas to access and feed on treated blood via an artificial feeding system. Each compound was tested by serial dilution to determine its minimum effective doses. Fleas were fed on treated blood for 24 h, after which the compound’s effect was recorded. Insecticidal activity was determined on the basis of the number of dead fleas recovered from the feeding system. Contact Test. This test was conducted as described by Wade et al. and Zakson-Aiken et al.64,65 In brief, adult fleas were distributed into wells of a microplate precoated with a serial dilution of the compounds to be evaluated for insecticidal activity. The fleas were left in contact with the compound for 24 h. Insecticidal activity was confirmed upon death of the adult fleas. In these assays, a compound needed to exhibit an insecticidal efficacy of >80% at a concentration of 100 ppm (100 μg/mL) to qualify for further testing. Activity in Vitro against R. sanguineus. Immersion Test. This test was conducted as described by Lovis et al.66 In brief, adult R. sanguineus were seeded into individual wells of a microtitration plate containing the test substances to be evaluated. Individual test compounds were tested by serial dilution to determine their minimum effective doses. Ticks were left in contact with the test compound for 10 min and then incubated at 28 °C and 80% relative humidity for 7 G
DOI: 10.1021/acs.organomet.6b00577 Organometallics XXXX, XXX, XXX−XXX
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Organometallics days, during which the test compounds’ effects were monitored. Acaricidal activity was confirmed on the basis of the pattern of lethality observed. Contact (Tarsal) Test. This test was conducted as described by Lovis et al.67 In brief, the test was performed by precoating wells of a 96-well microliter plate with a serial dilution of the compound, allowing the evaluation of antiparasitic activity by contact with ticks. Adult ticks were then distributed to individual wells of the plate and incubated at 28 °C and 80% relative humidity for 7 days, during which the test compound’s effect was monitored. Acaricidal activity was confirmed upon death of the adult ticks. Cell Culture. Human cervical carcinoma cells (HeLa) cells were cultured in DMEM (Gibco) supplemented with 5% fetal calf serum (FCS, Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C and 5% CO2. The normal human fetal lung fibroblast MRC-5 cell line was maintained in F-10 medium (Gibco) supplemented with 10% FCS (Gibco), penicillin (100 U/mL), and streptomycin (100 μg/ mL). Cytotoxicity Studies. Cytotoxicity studies were performed on two different cell lines, namely HeLa and MRC-5, by a fluorometric cell viability assay using Resazurin (Promocell GmbH). Briefly, 1 day before treatment, cells were seeded in triplicates in 96-well plates at a density of 4 × 103 cells/well for HeLa and 7 × 103 cells/well for MRC5 in 100 μL of growth medium. Upon treating cells with increasing concentrations of compounds for 48 h, the medium was removed, and 100 μL of complete medium containing Resazurin (0.2 mg/mL final concentration) was added. After 4 h of incubation at 37 °C, fluorescence of the highly red fluorescent product Resorufin was quantified at 590 nm emission with 540 nm excitation wavelength in a SpectraMax M5 microplate reader.
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Yourgene Bioscience, the Alexander von Humboldt Foundation, and The University of Melbourne. R.B.G. is a grateful recipient of a Professorial Humboldt Research Award. The authors thank Dr Jacques Bouvier (Novartis Animal Health, StAubin, Switzerland) and Dr. Noëlle Gauvry (Novartis Animal Health, Basel, Switzerland) for their help with the biological assays.
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ABBREVIATIONS: AADs, amino-acetonitrile derivatives; C. felis, Ctenocephalides felis; D. immitis, Dirofilaria immitis; ESI-MS, electrospray ionization-mass spectrometry; H. contortus, Haemonchus contortus; LDA, larval development assay; L. cuprina, Lucilia cuprina; nAChR, nicotinic acetylcholine receptor; o.n., overnight; P. falciparum, Plasmodium falciparum; ROS, reactive oxygen species; R. sanguineus, Rhipicephalus sanguineus; r.t., room temperature; SAR, structure−activity relationship; T. colubriformis, Trichostrongylus colubriformis
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.organomet.6b00577. Crystallographic data (CIF) Crystallographic data (CIF) Crystallographic data (CIF) 1 H, 13C, and 19F NMR spectra of 2−8, crystallographic data, UV traces of UPLC-ESI-MS, 1H−13C HMBC NMR spectra of 6 and 7, and antiparasitic activity against C. felis, L. cuprina, and R. sanguineus of organometallic precursors and derivatives 2−8 (PDF) Crystallographic data (CIF)
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REFERENCES
AUTHOR INFORMATION
Corresponding Authors
*R.B.G.: e-mail,
[email protected]; web, www.gasserlab. org; tel, +61 3 9731 2283. *G.G.: e-mail,
[email protected]; web, www. gassergroup.com; tel, +41 44 635 46 30. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was financially supported by the Swiss National Science Foundation (Professorship Nos. PP00P2_133568 and PP00P2_157545 to G.G.), the University of Zurich (G.G., S.F.), the Stiftung für wissenschaftliche Forschung of the University of Zurich (G.G., S.F.), the Novartis Jubilee Foundation (G.G.), and the Kurt u. Senta Hermann Stiftung (S.F.). R.B.G.’s research program is supported by the Australian Research Council (ARC), the National Health and Medical Research Council (NHMRC), Melbourne Water Corporation, H
DOI: 10.1021/acs.organomet.6b00577 Organometallics XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.organomet.6b00577 Organometallics XXXX, XXX, XXX−XXX
