Methoxy-derivatization of alkyl chains increases the in vivo efficacy of cationic Mn porphyrins. Synthesis, characterization, SOD-like activity, and SOD-deficient E. coli study of meta Mn(III) N-methoxyalkylpyridylporphyrins

Abstract

Cationic Mn(III) N-alkylpyridylporphyrins (MnPs) are potent SOD mimics and peroxynitrite scavengers and diminish oxidative stress in a variety of animal models of central nervous system (CNS) injuries, cancer, radiation, diabetes, etc. Recently, properties other than antioxidant potency, such as lipophilicity, size, shape, and bulkiness, which influence the bioavailability and the toxicity of MnPs, have been addressed as they affect their in vivo efficacy and therapeutic utility. Porphyrin bearing longer alkyl substituents at pyridyl ring, MnTnHex-2-PyP(5+), is more lipophilic, thus more efficacious in vivo, particularly in CNS injuries, than the shorter alkyl-chained analog, MnTE-2-PyP(5+). Its enhanced lipophilicity allows it to accumulate in mitochondria (relative to cytosol) and to cross the blood-brain barrier to a much higher extent than MnTE-2-PyP(5+). Mn(III) N-alkylpyridylporphyrins of longer alkyl chains, however, bear micellar character, and when used at higher levels, become toxic. Recently we showed that meta isomers are ∼10-fold more lipophilic than ortho species, which enhances their cellular accumulation, and thus reportedly compensates for their somewhat inferior SOD-like activity. Herein, we modified the alkyl chains of the lipophilic meta compound, MnTnHex-3-PyP(5+) via introduction of a methoxy group, to diminish its toxicity (and/or enhance its efficacy), while maintaining high SOD-like activity and lipophilicity. We compared the lipophilic Mn(III) meso-tetrakis(N-(6'-methoxyhexyl)pyridinium-3-yl)porphyrin, MnTMOHex-3-PyP(5+), to a hydrophilic Mn(III) meso-tetrakis(N-(2'-methoxyethyl)pyridinium-3-yl)porphyrin, MnTMOE-3-PyP(5+). The compounds were characterized by uv-vis spectroscopy, mass spectrometry, elemental analysis, electrochemistry, and ability to dismute O(2)˙(-). Also, the lipophilicity was characterized by thin-layer chromatographic retention factor, R(f). The SOD-like activities and metal-centered reduction potentials for the Mn(III)P/Mn(II)P redox couple were similar-to-identical to those of N-alkylpyridyl analogs: log k(cat) = 6.78, and E(1/2) = +68 mV vs. NHE (MnTMOHex-3-PyP(5+)), and log k(cat) = 6.72, and E(1/2) = +64 mV vs. NHE (MnTMOE-3-PyP(5+)). The compounds were tested in a superoxide-specific in vivo model: aerobic growth of SOD-deficient E. coli, JI132. Both MnTMOHex-3-PyP(5+) and MnTMOE-3-PyP(5+) were more efficacious than their alkyl analogs. MnTMOE-3-PyP(5+) is further significantly more efficacious than the most explored compound in vivo, MnTE-2-PyP(5+). Such a beneficial effect of MnTMOE-3-PyP(5+) on diminished toxicity, improved efficacy and transport across the cell wall may originate from the favorable interplay of the size, length of pyridyl substituents, rotational flexibility (the ortho isomer, MnTE-2-PyP(5+), is more rigid, while MnTMOE-3-PyP(5+) is a more flexible meta isomer), bulkiness and presence of oxygen.

Fig. 1

Mn porphyrins studied: MnTE-2-PyP⁵⁺, MnTE-3-PyP⁵⁺, MnTMOE-3-PyP⁵⁺, MnTnHex-2-PyP⁵⁺, MnTHex-3-PyP⁵⁺, and MnTMOHex-3-PyP⁵⁺.

Fig. 2

Synthesis of meta N-methoxyalkylpyridylporphyrins (H₂ TMOE-3-PyP⁴⁺ and H₂ TMOHex-3-PyP⁴⁺) and their Mn complexes (MnTMOE-3-PyP⁵⁺ and MnTMOHex-3-PyP⁵⁺).

Fig. 3

Comparison of the stability of MnPs towards oxidative degradation with H₂ O₂. Peroxide was produced in the interaction of MnPs with ascorbate.^,, The conditions were: 6 μM MnPs, 0.42 mM sodium ascorbate, pH 7.8 (Fig. 3A). The reduction of ortho MnPs, i.e. shift in Soret band, was observed upon the addition of ascorbate. The absorbance, A, of reduced Mn^IITE-2-PyP⁴⁺and Mn^IITnHex-2-PyP⁴⁺ was measured at 437 and 439 nm, respectively. With meta isomers, at the time scale of our spectrophotometer, only species with Mn in +3 oxidation state were observed, and their disappearance followed at 460 nm. At higher concentrations, i.e. 30 μM MnP and 3.3 mM ascorbate, the reduced species and its disappearance was visible (not shown). Fig. 3B clearly indicates the enhanced stability of MnTnHex-2-PyP⁵⁺ relative to its meta analog, MnTnHex-3-PyP⁵⁺ and shorter-chained ethyl species, MnTE-2-PyP⁵⁺, and much more so within a 2.5 h time frame and under the conditions indicated. A sharp drop in absorbance likely coincides with accumulated H₂ O₂ needed to degrade more resistant ortho porphyrins. As seen from a shorter time scale of 1.5 h (Fig. 3A), meta porphyrins aremore prone to oxidative degradation than ortho. Charges on porphyrins are omitted for simplicity.

Fig. 4

The lipophilicity of metal-free porphyrins and their ligands expressed here in terms of chromatographic retention factor, R_f. The R_f values are linearly related to log P_OW values. The small differences in R_f values translate into large differences in log P_OW values. Lipophilicity of Mn complexes is lower than of their metal-free ligands due to higher solvation of the metal site. The effect is more drastic with porphyrins bearing shorter substituents. Longer-chained analogs, alkyl and methoxyalkyl are more lipophilic than their shorter-chained analogs. Introduction of a methoxy group reduces lipophilicity of longer alkyl analog, H₂ TnHex-3-PyP⁴⁺ and its Mn complex. Charges on porphyrins are omitted for simplicity.

Fig. 5

The differential effect of the MnPs on the aerobic growth of SOD-deficient E. coli, JI in restricted five amino acid medium: impact of oxygen. The turbidity of medium was measured by absorbance at 600 nm, and is proportional to the E. coli growth. MnPs were given at 1–60 μM. The growth of JI and AB alone are shown also. Without cytosolic SOD enzymes or their mimics, SOD-deficient E. coli grows very poorly in a restricted medium. An SOD mimic substitutes for the lack of cytosolic SODs in the SOD-deficient strain JI. The magnitude of its effect on the E. coli growth depends upon its efficacy and ability to enter the E. coli cell. The ortho Mn porphyrin, MnTE-2-PyP⁵⁺, is routinely used as an internal control in all our studies to account for the differences in the growth of E. coli. In numerous experiments MnTE-2-PyP⁵⁺ at 20 μM allowed the full growth of SOD-deficient E. coli. Since the meta isomeric N-methoxyalkylpyridylporphyrins are explored in this study, to witness their advantage over meta alkylpyridylporphyrins, meta MnTE-3-PyP⁵⁺ and meta MnTnHex-3-PyP⁵⁺ were tested also. When compounds enter the cell and are potent SOD mimics also, they exert efficacy at very low 1 and 5 μM concentrations. Such was the case with both MnTMOE-3-PyP⁵⁺ and MnTMOHex-3-PyP⁵⁺. Yet, when used at higher concentrations, MnTMOHex-3PyP⁵⁺ starts exerting toxicity, while MnTMOE-3-PyP⁵⁺ was fully efficacious up to 60 μM levels. Analogous hexylpyridylporphyrin, MnTnHex-3-PyP⁵⁺, is much more toxic: no improvement in the growth of JI was seen at 1 μM concentration, and full suppression of its growth was observed at 5 μM. The ortho analogue, MnTnHex-2-PyP⁵⁺ is slightly less toxic than meta MnTnHex-3-PyP⁵⁺ as it is bulkier and less lipophilic. In five independent experiments, the methoxyethyl analog MnTMOE-3-PyP⁵⁺ was consistently the most efficacious compound: it allows SOD-deficient E coli to grow better than wild type AB, and better than both ortho and meta ethyl analogues, MnTE-2(or 3)-PyP⁵⁺. Such a remarkable effect of MnTMOE-3-PyP⁵⁺ may be the consequence of the favorable interplay of lipophilicity, size, shape, rotational flexibility, and presence of oxygen. The inset shows the effect of MnPs at two concentrations and at 24 h. At 5 μM both methoxyalkylpyridylporphyrins are superior to the alkylpyridyl analogs tested; i.e. they help SOD-deficient E. coli (JI) to grow to a similar extent as does wild type strain (AB). Taken together, the data indicate that introduction of oxygens into the chains makes alkylpyridylporphyrins less toxic and more efficacious. See the Results and discussion section for possible explanation of such effects. The charges on Mn porphyrins are omitted for simplicity.