Article pubs.acs.org/Organometallics
Antibacterial and Antiparasitic Activity of Manganese(I) Tricarbonyl Complexes with Ketoconazole, Miconazole, and Clotrimazole Ligands Peter V. Simpson,*,†,§ Christoph Nagel,† Heike Bruhn,‡ and Ulrich Schatzschneider*,† †
Institut für Anorganische Chemie, Julius-Maximilians-Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany Institut für Molekulare Infektionsbiologie, Julius-Maximilians-Universität Würzburg, Josef-Schneider-Strasse 2/D15, D-97080 Würzburg, Germany
‡
S Supporting Information *
ABSTRACT: Five manganese(I) tricarbonyl complexes of the general formula [Mn(CO)3(bpyR,R)(azole)]PF6 with R = H, COOCH3, and azole = ketoconazole (ktz), miconazole (mcz), and clotrimazole (ctz) were synthesized and fully charaterized, including X-ray structure analysis for the ctz compound. The antibacterial activity on a panel of eight Gram-positive and Gram-negative bacterial strains was determined. While there was no effect on the latter microorganisms, the ctz complex showed submicromolar activity on Staphylococcus aureus and S. epidermidis with MIC values of 0.625 μM. Antiparasitic activity was investigated on Leishmania major and Trypanosoma brucei. Coordination of the organic azole drugs to the Mn(CO)3 moiety led to complexes with low micromolar IC50 values, but their potential for antileishmanial therapy is low due to comparable toxicity on mammalian cell lines 293T and J774.1. In contrast, the antitrypanosomal activity is much more promising, and the most potent compound incorparting the ktz ligand has an IC50 value on T. brucei of 0.7 μM with selectivity on parasitic over mammalian cells as expressed by a selectivity index above 10. These results demonstrate that metal coordination of established drugs can significantly improve their biological activity and expand their range of medicinal applications.
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INTRODUCTION Anticancer-active metal complexes are well established in medicinal inorganic chemistry today, with cisplatin derivatives as a mainstay of chemotherapeutic protocols in current clinical use.1 However, although increasing bacterial resistance to established antibiotics2 and very limited treatment options for neglected tropical diseases3,4 call for the development of new lead compounds, metal complexes have not been explored to their full potential in this context so far.5 The compounds studied for antibacterial or antiparasitic activity are often based on relatively weak metal−ligand interactions and insufficiently characterized with regard to their speciation in complex biological media. The most notable exception is ferroquine, an organometallic antimalarial drug candidate in advanced clinical trials (Chart 1).6−9 Only very recently have other organometal complexes also emerged as quite potent agents against pathogenic bacteria10 and parasitic microorganisms.11,12 In particular, various organometallic gold compounds13 as well as ruthenium(II) complexes14 have received significant attention due to facile structural variation important for structure−activity relationship studies. Also, thiosemicarbazone metal complexes are at the focus of a number of publications.15−17 Other metal−ligand architectures have been much less studied to date. In our own group, cymantrene− chloroquine conjugates as well as rhodium(I) and iridium(I) N© XXXX American Chemical Society
Chart 1. Some Metal Complexes for (Top Left) Clinical Use in Anticancer Chemotherapy and (Top Center and Right, Bottom) Antibacterial and Antiparasitic Activity Studies
heterocyclic carbene (NHC) complexes incorporating bulky, lipophilic cationic groups were recently explored for their antibacterial and antiparasitic potency (Chart 1).18,19 Although some promising activity was found, in particular on trypanosomes as the causative agent of African sleeping sickness, low selectivity on pathogenic over mammalian cells was a major problem. Received: May 28, 2015
A
DOI: 10.1021/acs.organomet.5b00458 Organometallics XXXX, XXX, XXX−XXX
Article
Organometallics
metrical stretching A″ and the CO stretching mode of the carbonyl group trans to the axial ligand A′(2) are observed at lower wavenumbers. These values are summarized in Table 1.
In addition to the development of totally new metal-based lead structures to tackle this problem, current approaches are also based on the coordination of well-established drugs and drug candidates to inert organometal fragments. This strategy raises the interesting question of how metal binding will modulate the biological activity of the drug and if synergistic effects can be achieved this way.20,21 Particularly promising in this context are azole-based drugs, which have been explored in the context of antifungal chemotherapy for a number of decades.22−24 Due to the presence of free nitrogen donor centers, these compounds can easily be coodinated to a wide range of metal-coligand fragments. So far, most of the published work has been based on ruthenium(II) arene complexes.25,26 In contrast, no metal carbonyl compounds have been explored in this context so far. Therefore, it was the aim of the present work to explore the impact of azole (ketoconazole, miconazole, and clotrimazole) drug coordination to a very stable Mn(CO)3(N−N) moiety on the biological activity of the resulting compounds against a panel of pathogenic bacteria and parasitic microorganisms27 responsible for neglected diseases.
Table 1. Characteristic IR Carbonyl Stretching Bands for 3− 7 A′(1) in cm−1
A″ in cm−1
A′(2) in cm−1
3 4 5 6 7
2035 2036 2033 2036 2038
1946 1948 1940 1948 1959
1927 1930 1927 1932 1928
Complexes 4 and 6 exhibit slightly higher energy carbonyl stretching bands compared to 3 and 5, consistent with the expected effect of the methyl ester-functionalized versus the nonfunctionalized bipyridine ligand on the electron density at the metal center. Complex 7, however, displays the highest energy carbonyl bands, perhaps due to the electron-withdrawing effect of the triphenylmethane group. All complexes exhibit the expected signals in the 1H and 13C NMR spectra indicative of symmetrical bpy coordination, with only one set of signals present for the two halves of the ligand. The bipyridine H6 signals appear in a range of 9.26−9.56 ppm. A trend similar to the IR data is observed, with the signals of the methyl ester-functionalized complexes 4 and 6 shifted by about 0.24 ppm to lower field compared to 3 and 5. The imidazolyl H2 signal is observed at 7.48−7.65 ppm in 3−6. For 7, the latter signal is shifted upfield to 6.58 ppm, suggesting partial shielding by one or more of the phenyl substituents, which are spatially closer than in the other complexes. Signals in the 13C NMR spectra associated with the carbonyl ligands are found at ca. 219 and 218 ppm for the equatorial and axial carbonyl groups, respectively. Characteristic [M − PF6]+ and [M − 3CO − PF6]+ fragments were the dominant species observed for all complexes by ESI mass spectrometry. Crystal Structure. The clotrimazole complex 7 was crystallized by diffusion of diethyl ether into a solution of the compound in acetonitrile at −20 °C. X-ray structure analysis shows the manganese atom in a pseudooctahedral environment with the fac-Mn(CO)3 moiety coordinated by the bidentate bpy ligand in a symmetrical way and the azole ligand bound via the imidazole N(1) atom (Figure 1). The metrical parameters around the metal center compare very well with those recently reported for the parent compound incorporating N-phenylimidazole.30,31 The five-membered ring of the ctz ligand is somewhat tilted toward the bpy ring plane with an angle of only 75.3° instead of the expected perpendicular arrangement. Furthermore, it is in an almost eclipsed conformation relative to one of the N−Mn−CO vectors, with a N(4)−Mn(1)−N(1)− C(4) torsion angle of only 21.1°. The central C(17) carbon atom of the ctz ligand shows the expected metrics of an sp3hybridized center with angles in the range 107.7 to 112.9°. The chloro-substituted phenyl ring is tilted toward one-half of the bpy ligand, but with the shortest Cl(1)−C(8) distance at 3.84 Å; this seems to be more dictated by the general packing than a chloride−pyridine interaction. The same also applies to the second half of the bpy, which is more or less coplanar to the C(25)-to-C(29) phenyl ring at a 28.7° angle, but with a centroid−centroid distance of 4.79 Å, πstacking interactions are not operative. In addition to clotrimazole itself,32 structural data for a number of transition
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RESULTS AND DISCUSSION Synthesis and Spectroscopic Studies. Treatment of manganese(I) tricarbonyl complexes 1 and 2 with silver triflate in acetone at room temperature for bromide abstraction and subsequent addition of azole ligands ketoconazole (ktz), miconazole (mcz), and clotrimazole (ctz) led to the formation of [Mn(CO)3(bpy)(azole)]OTf as oily residues. Precipitation of the triflate salts with ammonium hexafluorophosphate in methanol/water mixtures followed by column chromatography on silica gel using a dichloromethane/methanol mixture as the eluent gave 3−7 as air- and moisture-stable complexes in moderate to good yield (Scheme 1). The complexes show excellent solubility in common organic solvents such as chloroform, dichloromethane, acetone, dimethyl sulfoxide, and dimethylformamide. The IR spectra of 3−7 display the characteristic three carbonyl stretching bands as expected from the local Cs point group. In line with the work of Vrieze28 as well as our own recent DFT calculations,29 the highest energy band is assigned to the symmetrical stretching mode A′(1), while the antisymScheme 1. Synthesis of Metal Tricarbonyl Complexes 3−7 and Structure of Azole Ligands ktz, mcz, and ctza
a
compound
The metal-coordinated atom is highlighted in color. B
DOI: 10.1021/acs.organomet.5b00458 Organometallics XXXX, XXX, XXX−XXX
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Organometallics
In line with previous data,18,19 essentially all tested compounds were completely inactive on the Gram-negative bacteria (Table 2). The only exception is complex 5, which has very moderate MIC values of 10−20 μM on E. coli, Y. pseudotuberculosa, and Y. pestis. Interestingly, the metal compound still has a higher activity than the free mcz ligand, with MIC > 40 μM. In contrast, the title compounds turned out to be quite active on the Gram-positive bacteria, where low to submicromolar MIC values were determined for most of the metal complexes (Table 2). A number of general trends emerged from relation of their potency to structural features. With only a very few exceptions, metal complexes 3−7 were more active than the purely organic compounds ktz, mcz, and ctz, up to a factor of 30 in the case of 7 versus ctz, with MIC values of 0.625 and 20 μM, respectively. Among the metal complexes, the activity increases with the type of azole ligand in the order ktz < mcz < ctz. Introduction of the methyl ester group in the 4- and 4′-position of the 2,2′bipyridine ligand in 3 versus 4 and 5 versus 6 generally resulted in an increase of the MIC value by a factor of 2−16 and thus lower potency. The effect was less pronounced in the mcz compared to the ktz compounds. As a general trend, the activity increases with a decrease of the molecular weight, with ctz compound 7 showing the highest potency and MIC values in the range of 0.625 to 2.5 μM on all four bacterial strains tested. Finally, when comparing the different species, the staphylococci were somewhat more responsive than the enterococci for most compounds. The highest activity was observed for [Mn(CO)3(bpy)(ctz)]PF6 (7), which has a submicromolar MIC value of 0.625 μM on both S. aureus and S. epidermidis. This compares favorably with the NHC rhodium and iridium complexes recently tested in our group on the same panel of bacteria,19 where the most potent compound had a MIC values of 2.5 μM. Antiparasitic Activity. Although originally developed as antifungal agents, azole compounds have also been explored for activity on kinetoplastids such as Leishmania since these parasites also require ergosterol for their metabolism and share this biosynthetic pathway with fungi. The mechanism of action is based on inhibition of lanosterol demethylase, which leads to accumulation of 14α-methyl sterols.4 To explore the antiparasitic potential of the title compounds, the antileishmanial and antitrypanosomal activity of 3−7 as well as ktz, mcz, and ctz was determined on Leishmania major and Trypanosoma brucei using the Alamar Blue assay.18 In addition, IC50 values were also determined on 293T human embryonal kidney cells as well as J774.1 murine macrophages to calculate the selectivity
Figure 1. Molecular structure of the cation of 7. Atomic displacement ellipsoids are shown at the 50% probability level. The hexafluorophosphate counterion and a cocrystallized acetonitrile solvent molecule are omitted for clarity. Selected bond lengths (Å): Mn(1)−C(1), 1.818(5); Mn(1)−C(2), 1.806(5); Mn(1)−C(3), 1.810(5); Mn(1)−N(1), 2.070(3); Mn(1)−N(3), 2.036(4); Mn(1)− N(4), 2.044(3); C(1)−O(1), 1.145(5); C(2)−O(2), 1.152(5); C(3)− O(3), 1.157(5).
metal complexes with ctz as a ligand have been reported in the literature. While most of the work so far has focused on coordination compounds of copper,33−35 zinc,35 palladium,36 cadmium,37 and gold,38 there are also some studies on bioorganometallic ctz compounds with the azole bound to a RuII(arene) or RhI(cod) fragment.25,26,38 However, to the best of our knowledge, no crystallographic data has been reported so far on clotrimazole coordinated to a metal−carbonyl moiety. Structural data on miconazole compounds are much more scarce. In addition to the purely organic drug,39,40 only one report on cobalt and copper complexes with mcz ligands has appeared in the literature so far.41 The same also applies to ketoconazole,42,43 with a RuII(arene)(ktz) compound as the only metal complex thereof crystallographically characterized to date.20 Antibacterial Activity. Since azole-based drug candidates have found widespread interest in medicinal chemistry,22−24,44,45 we tested the biological activity of manganese tricarbonyl complexes 3−7 as well as the organic ligands ctz, ktz, and mcz on a panel of eight different bacterial strains, Gram-positive Staphylococcus aureus, S. epidermidis, Enterococcus faekalis, and E. faecium, and Gram-negative Escherichia coli, Pseudomonas aeruginosa, Yersinia pseudotuberculosa, and Y. pestis.
Table 2. Minimum Inhibitory Concentration (MIC) in μM for Complexes 3−7 and Ketoconazole (ktz), Miconazole (mcz), and Clotrimazole (ctz) on a Panel of Eight Different Bacterial Strainsa Gram-positive bacteria
Gram-negative bacteria
compound
S. aureus
S. epidermidis
E. faekalis
E. faecium
E. coli
P. aeruginosa
Y. pseudotuberculosa
Y. pestis
3 4 5 6 7 ktz mcz ctz
2.5 40 1.25 2.5 0.625 40 5 20
2.5 20 1.25 2.5 0.625 40 2.5 2.5
20 >40 2.5 10 2.5 >40 10 40
10 >40 1.25 5 2.5 >40 10 20
>40 >40 20 >40 40 >40 >40 >40
>40 >40 >40 >40 >40 >40 >40 >40
>40 >40 20 >40 >40 >40 >40 >40
>40 >40 10 >40 40 >40 >40 >40
a
Full names: Staphylococcus aureus, Staphylococcus epidermis, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Pseudomonas aeruginosa, Yersinia pseudotuberculosa, Yersinia pestis. C
DOI: 10.1021/acs.organomet.5b00458 Organometallics XXXX, XXX, XXX−XXX
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Organometallics
Table 3. IC50 Values in μM for Complexes 3−7 and Ketoconazole (ktz), Miconazole (mcz), and Clotrimazole (ctz) toward L. major and T. brucei Together with Data on Mammalian Cell Lines 293T and J774.1a compound 3 4 5 6 7 ktz mcz ctz
293T 6.3 ± 13.3 ± 1.9 ± 13.8 ±
