Orally available Mn porphyrins with superoxide dismutase and catalase activities

Abstract

Superoxide dismutase/catalase mimetics, such as salen Mn complexes and certain metalloporphyrins, catalytically neutralize reactive oxygen and nitrogen species, which have been implicated in the pathogenesis of many serious diseases. Both classes of mimetic are protective in animal models of oxidative stress. However, only AEOL11207 and EUK-418, two uncharged Mn porphyrins, have been shown to be orally bioavailable. In this study, EUK-418 and several new analogs (the EUK-400 series) were synthesized and shown to exhibit superoxide dismutase, catalase, and peroxidase activities in vitro. Some also protected PC12 cells against staurosporine-induced cell death. All EUK-400 compounds were stable in simulated gastric fluid, and most were substantially more lipophilic than the salen Mn complexes EUK-189 and EUK-207, which lack oral activity. Pharmacokinetics studies demonstrate the presence of all EUK-400 series compounds in the plasma of rats after oral administration. These EUK-400 series compounds are potential oral therapeutic agents for cellular damage caused by oxidative stress.

Scheme 1

Synthesis of 5, 15 porphyrin ligands for all EUK-400 compounds except for EUK-425. TFA is trifluoroacetic acid and DDQ is 2,3-dichloro-5,6-dicyanobenzoquinone. Further details are described in Materials and methods

Scheme 2

Incorporation of metal to form Mn porphyrin complex for all EUK-400 compounds except for EUK-425. Further details are described in Materials and methods

Fig. 1

a Structures of EUK-400 series compounds. Structures are also given for the related Mn porphyrins 5,15-bis(methoxycarbonyl)-10,20-bistrifluoromethylporphyrinato manganese(III) chloride (AEOL11207) [27], manganese(III) tetrakis(4-benzoic acid) porphyrin (MnTBAP), and manganese(III) tetrakis(1-methyl-4-pyridyl) porphyrin (MnTMPyP) [5]. b Structures of the salen Mn complexes EUK-189 and EUK-207. OAc acetoxy (CH₃COO)

Scheme 3

Synthesis of EUK-425, following the method of Meunier and Cosledan [29]

Fig. 2

Some EUK-400 series compounds protect PC12 cells against staurosporine (STS)-induced cell death. Rat pheochromocytoma (PC12) cells were cultured in collagen-coated 96-well plates and STS (5 μM) was added to induce cell death. Test compounds were added together with the STS. After an 18–24-h incubation, test medium was removed, and cell viability was determined using the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide viability assay, as described in “Materials and methods.” Control cells received no STS. In the absence of STS, the EUK-400 series compounds showed cytotoxicity at concentrations of 10–30 μM. The data shown are direct comparisons of the compounds using means of duplicates, and error bars indicate the range. These data are representative of at least two additional independent tests of each compound in triplicate

Fig. 3

In vivo oral availability of EUK-425 in rats. EUK-425 was administered to Sprague–Dawley rats (n = 3) by intragastric gavage (PO) or intravenous injection (IV). The compound was formulated in dimethyl sulfoxide/Solutol HS 15/phosphate-buffered saline, pH 7.4 (5:5:90, by volume), and the doses were 5 mg kg⁻¹ for PO and 1 mg kg⁻¹ for IV administration. The final volumes administered were 1.35–1.53 ml. Plasma was collected from a jugular vein catheter at various time points, and quantitation of compounds was performed by liquid chromatography with tandem mass spectroscopy. Fifty microliters of plasma was precipitated with 200 μl acetonitrile, and the supernatant was analyzed using a TSQ Quantum machine. Ten-microliter aliquots were injected for analysis. The high performance liquid chromatography column was Synergi Fusion-RP 80, 4 μm (2 mm × 50 mm). The runs were performed at a flow rate of 0.5 ml min⁻¹ with mobile phase A consisting of 13.3 mM ammonium formate, 6.7 mM formic acid in water. Mobile phase B was 6 mM ammonium formate, 3 mM formic acid in water/acetonitrile (1:9 v/v). The gradient was 20–100% mobile phase B over 2.5 min. The apparent molecular ion for EUK-425 was 516.0 m/z → 475.2 m/z