Evaluation of the antiparasitic activities of imidazol‐2‐ylidene–gold(I) complexes

A series of cationic gold(I)–carbene complexes with various 4,5‐diarylimidazolylidene ligands were either newly prepared or repurposed for testing against protozoal Leishmania major, Toxoplasma gondii, and Trypanosoma brucei parasites. The syntheses of the new complexes 1b and 1c were described. Ferrocene compound 1a showed the highest activities against L. major amastigotes and T. gondii and distinct selectivity for T. gondii cells when compared with the activity against nonmalignant Vero cells. The ferrocene derivatives 1a–c are generally more active against the L. major amastigotes and the T. gondii tachyzoites than the other tested anisyl gold complexes and the approved drugs atovaquone and amphotericin B. Compounds 1a and 1e showed the highest selectivities for L. major amastigotes. Compounds 1d and 1f showed the highest selectivities for L. major promastigotes; 1f was the most active compound against L. major promastigotes of this series of compounds. The 3,4,5‐trimethoxyphenyl analog 1b also exhibited a much greater selectivity for T. b. brucei cells when compared with its activity against human HeLa cells.

ferrocene derivatives have previously shown activities against various parasites. [13][14][15] In addition, imidazoles, on their own, also displayed distinct antimicrobial and antiparasitic activities. [16,17] We now evaluated the scope and structure dependence of the antiparasitic effects of a series of known and new gold(I)-NHC complexes of our lab on the protozoal parasites Leishmania major, T. brucei (both kinetoplastid parasites), and Toxoplasma gondii (apicomplexan parasite). Some of the known gold complexes used in this study have already shown in vivo activity against tumor xenografts with good tolerability by the laboratory animals and, thus, these complexes are suitable for repurposing against parasites. [18,19] 2 | RESULTS AND DISCUSSION The known complexes 1a and 1d-g were prepared according to literature procedures (Figure 1). [18,19] The new complexes 1b and 1c were prepared accordingly and tested to assess the influence of methoxy substituents on the activity against and the selectivity for protozoal parasites (Scheme 1). The reaction of ferrocenecarboxaldehyde with ethyl amine and TosMIC reagents 2b and 2c, respectively, afforded the N-ethyl-imidazoles 3b and 3c in good yields. High yield alkylation with ethyl iodide was followed by quantitative conversion of the iodides 4b and 4c to the BF 4 salts 5b and 5c. Finally, reaction of 5b and 5c with Ag 2 O and transmetallation with 0.5 equiv.
Au(DMS)Cl led to the target complexes 1b and 1c as brown solids in good yields.
The complexes 1a-g ( Figure 1) were initially tested for their activity against T. gondii tachyzoites ( Table 1). The ferrocene derivatives  were also more active than the positive control atovaquone (ATO), which is an approved drug for the treatment of toxoplasmosis.
The activity of complexes 1a-g against L. major promastigotes and amastigotes was also determined ( Table 2). The ferrocenes 1a-c were the most efficient growth inhibitors of the L. major amastigotes with complex 1a showing the highest activity (EC 50 = 0.11 µM) and a slight selectivity for the amastigotes (SI = 3.32). However, anisyl-NHC complex 1e, while being the second least active compound against amastigotes, showed the highest selectivity for them (SI = 12.8). The anisyl-NHC complexes 1d and 1f were slightly more active than the ferrocenes against L. major promastigotes and less active against the amastigotes. For approved antileishmanial drugs, a high activity against amastigotes was observed and other drug candidates also showed higher activity against L. major amastigotes than against promastigotes. [20,21] When compared with the positive control amphotericin B (AmB), complexes 1a-d showed higher activities both against the promastigotes and against the amastigotes. In addition, compound 1f was more active than AmB against the promastigotes, and 1g against the amastigotes. Complex 1e showed virtually the same activity as AmB against amastigotes and considerable selectivity.
Compounds 1a and 1e were already described by our groups as antitrypanosomal compounds. [12] Hence, the new ferrocenes 1b and 1c, which are close analogs of 1a, were selected and also tested for their trypanocidal activity against bloodstream-form T.
b. brucei parasites by the Alamar Blue (AB) assay. The obtained results were compared with those previously observed for 1a and 1e (Table 3). [12] In particular, the new complex 1b exhibited high activity against T. b. brucei (IC 50 = 5 nM) and a high selectivity for

| General
All starting compounds were purchased from Aldrich. The known complexes 1a and 1d-g and the TosMIC reagents 2b and 2c were prepared according to literature procedures. [18,19,22] The analytical data of these compounds were in agreement with the published data.
The following instruments were applied for this study: melting points (uncorrected), Gallenkamp; infrared (IR) spectra, Perkin-Elmer Spectrum One FT-IR spectrophotometer with ATR-sampling unit; nuclear magnetic resonance spectra, Bruker Avance 300 spectro-

| Trypanosoma cell line and culture conditions
Cultivation of the T. b. brucei bloodstream-form cell strain Lister 427 was carried out in HMI-9 medium, pH 7.5, supplemented with 10% FBS at 37°C in a humidified 5% CO 2 atmosphere. [25]

| Alamar Blue (AB) assay
Viable cells after treatment with drug candidates were identified via the AB assay. [26][27][28][29] Pink resorufin is formed in intact cells from the irreversible reaction of the blue dye resazurin and NADH.  [30,31]