Lipophilicity of potent porphyrin-based antioxidants. Comparison of ortho and meta isomers of Mn(III) N-alkylpyridylporphyrins

Abstract

Mn(III) N-alkylpyridylporphyrins are among the most potent known SOD mimics and catalytic peroxynitrite scavengers, and modulators of redox-based cellular transcriptional activity. In addition to their intrinsic antioxidant capacity, bioavailability plays major role in their in vivo efficacy. While of identical antioxidant capacity, lipophilic MnTnHex-2-PyP is up to 120-fold more efficient in reducing oxidative stress injuries than hydrophilic MnTE-2-PyP. Due to limitations of analytical nature, porphyrin lipophilicity has been often estimated by thin-layer chromatographic R_f parameter, instead of the standard n-octanol/water partition coefficient, P_OW. Herein we used a new methodological approach to finally describe the MnP lipophilicity, by the conventional log P_OW means, for a series of biologically active ortho and meta isomers of Mn(III) N-alkylpyridylporphyrins. Three new porphyrins (MnTnBu-3-PyP, MnTnHex-3-PyP and MnTnHep-2-PyP) were synthesized to strengthen the conclusions. The log P_OW was linearly related to R_f and to the number of carbons in the alkyl chain (nC) for both isomer series; the meta isomers being 10-fold more lipophilic than the analogous ortho porphyrins. Increasing the length of the alkyl chain for 1 carbon atom increases the log P_OW value ~ 1 log unit with both isomers. Dramatic ~4 and ~5 orders of magnitude increase in lipophilicity of ortho isomers by extending pyridyl alkyl chains from 2 (MnTE-2-PyP, log P_OW = −6.25) to 6 (MnTnHex-2-PyP, log P_OW = −2.29) and 8 carbon atoms (MnTnOct-2-PyP, log P_OW = −0.77) parallels the increased efficacy in several oxidative-stress injury models, particularly those of the central nervous system where transport across the blood-brain barrier is critical. Although meta isomers are only slightly less potent SOD mimics and antioxidants than their ortho analogues, their higher lipophilicity and smaller bulkiness may lead to a higher cellular uptake and overall similar effectiveness in vivo.

Introduction

Oxidative stress is a common state in most pathological conditions such as cancer, diabetes, radiation injury, disorders of central nervous system (Alzheimer’s, Parkinson’s, multiple sclerosis, amyotrophic lateral sclerosis, stroke, spinal cord injury, chronic morphine tolerance), etc [1]. Thus synthetic antioxidants such as metal complexes (Mn salens [2], Mn cyclic polyamines [3], Mn porphyrins (MnP) [4]) and nitroxides [5] have been developed. Given the key role of mitochondria, compounds that accumulate there, such as MitoQ [6, 7] and Mn porphyrins [8] are of particular interest.

We have developed metalloporphyrin-based compounds that are potent mimics of superoxide dismutase based on structure-activity relationships (SAR) between the ability of MnPs to catalytically remove superoxide and the metal-centered redox potential for the Mn^IIIP/Mn^IIP redox couple, E_1/2 [9, 10]. The ability of Mn porphyrins to scavenge superoxide parallels their ability to catalytically remove peroxynitrite, a more harmful and powerful oxidant in vivo [11]. Levels of O₂^·− and ONOO⁻ and the other species that they generate (OH^·, H₂O₂, CO₃^·−, NO₂^·) are increased under pathological conditions and damage biological molecules leading to their loss of function. Such species also upregulate cellular transcription, propagating oxidative stress and, thus, the related disease condition. Indeed we have found that Mn porphyrins inhibit activation of major transcription factors, AP-1 [12], NF-κB [13,14] and HIF-1α [15].

The Mn(III) ortho meso-tetrakis-N-ethylpyridylporphyrin, MnTE-2-PyP is the prototypical MnP-based SOD mimic/peroxynitrite scavenger most studied in vivo [16 – 25]. Its efficacy in vivo is at least in part due to its accumulation in mitochondria [8] and, despite its overall 5+ charge, its pharmacokinetics in the mouse shows that the drug distributes into major tissues and organs, including the brain [15].

The most efficacious compounds with high k_cat, Mn(III) ortho meso-tetrakis-N-alkylpyridylporphyrins, have positively charged groups in ortho positions, in close vicinity to the metal site. Such design affords thermodynamic and electrostatic facilitation for the approach of negatively charged superoxide to the Mn center [10]. Recent studies have strongly supported the assumption that, in addition to high antioxidant potency, MnP bioavailability plays a major role, particularly in central nervous system disorders [26]. A MnTE-2-PyP analogue of equivalent antioxidant potency has been designed and synthesized with longer alkyl chains to yield an intuitively more lipophilic derivative, i.e. MnTnHex-2-PyP (Figure 1) [9]. Indeed, we have shown that MnTnHex-2-PyP is more effective in facilitating aerobic growth of SOD-deficient E. coli than its ethyl analogue (due at least in part to the fact that it accumulates in E. coli better [27]), thus offering protection at > 2 orders of magnitude lower concentration than MnTE-2-PyP [27]. The SOD-deficient E. coli that grew in the presence of MnTnHex-2-PyP has significantly higher ability to dismute O₂^·− than if E. coli grew in the presence of MnTE-2-PyP [27]. In animal models, MnTnHex-2-PyP was up to 120-fold more effective than MnTE-2-PyP (see Discussion also) at lowest therapeutically relevant doses (0.05 mg/kg) than any synthetic antioxidant thus far reported ^[1–4]. In rats, MnTnHex-2-PyP distributes between blood and brain at an 8:1 ratio 30 min after iv injection, while the corresponding ratio is 100:1 with MnTE-2-PyP [13, 15] ^[2]). In cerebral palsy model^[3] preliminary data show that only MnTnHex-2-PyP, but not MnTE-2-PyP, was effective when given to pregnant rabbit dam twice (intravenously), before and after uterus ischemia at only 0.05 mg/kg (0.6 mg total dose per rabbit dam). The efficacy has been ascribed to the improved ability of MnTnHex-2-PyP to cross several lipid barriers before entering fetal brain ^[3]. While both MnTE-2-PyP and MnTnHex-2-PyP are promising for clinical development, the latter is advantageous for central nervous system (CNS) injuries due to its higher ability to cross the blood-brain barrier. To note, both MnTE-2-PyP and MnTnHex-2-PyP are extremely stable and do not lose redox-active Mn site in the presence of other ligands (EDTA). No loss of MnTnHex-2-PyP happens within 3 months in 12 M HCl and only 50% of MnTE-2-PyP was lost within a month [9].

Figure 1.

Structures of ortho and meta isomers of Mn(III) N-alkylpyridylporphyrins.

Whereas the lipophilicity of MnPs seems to be a critical parameter for their action in vivo, it is often estimated in terms of the thin-layer chromatographic (TLC) retention factor values (R_f), which is not a common pharmaceutical parameter of drug lipophilicity and is meaningless for medical and pharmaceutical audiences, as it precludes the comparison with other drugs targeting similar diseases. A more familiar lipophilicity descriptor is the n-octanol/water partition coefficients (P_OW). However, MnPs distribute into n-octanol at extremely low levels, which imposes difficulties of analytical nature in determining log P_OW. Herein we have adapted a recent strategy [28] to determine the log P_OW of ortho and meta isomeric Mn porphyrins, and show that actual log P_OW, whose measurements are cumbersome, can be quickly and easily predicted based upon simple and straightforward R_f determinations.

Methods

General

Xanthine and equine ferricytochrome c (lot no. 7752) were from Sigma, whereas xanthine oxidase was prepared by R. Wiley [29] and was a gift from K.V. Rajagopalan. MnCl₂·4H₂O was purchased from J. T. Baker. Anhydrous N,N-dimethylformamide (DMF, Sigma-Aldrich Chemical Co) was kept over 4 Å molecular sieves. Ethyl ether (anhydrous), acetone, chloroform, and aluminum-backed silica gel 60 F254 TLC sheets were from EMD. NH₄PF₆ (>99.9999% purity) was from Advance Research Chemicals, Inc. KNO₃ and methanol were from Mallinckrodt. Acetonitrile was from Fisher Scientific. Plastic-backed silica gel TLC plates (Z122777-25EA, Batch#3110), tetrabutylammonium chloride and ethyl p-toluenesulfonate were purchased from Sigma-Aldrich. The n-butyl, n-heptyl and n-hexyl esters of p-toluenesulfonic acid were purchased from TCI America. All other chemicals and solvents were of analytical grade and used without further purification.

Electrospray ionization mass spectrometric analyses (ESI-MS) were performed as described elsewhere [30] on an Applied Biosystems MDS Sciex 3200 Q Trap LC/MS/MS spectrometer at Duke Comprehensive Cancer Center, Shared Resource PK Labs; all samples of ~5 μM concentration were prepared in a H₂O-acetonitrile (1:1, v/v; containing 0.1 % v/v HFBA) mixture and infused for 1 min at 10 μL/min into the spectrometer (curtain gas = 20 V; ion spray voltage = 3500 V; ion source = 30 V; T = 300 °C; declustering potential = 20 V; entrance potential = 1 V; collision energy = 5 V; gas = N₂).

UV/vis spectra were recorded in H₂O at room temperature on a Shimadzu UV-2501PC spectrophotometer with 0.5 nm resolution.

Elemental analyses were done by Atlantic MicroLab, Norcross, GA.

Porphyins

Caution needs to be exercised with all chemicals used in the synthesis including commercial porphyrins as their quality is variable [30]. The synthesis and characterization of all ortho isomers other than the n-heptyl analogue, which is given below, have been reported elsewhere [9]. The preparation of the other new compounds MnTnBu-3-PyP and MnTnHex-3-PyP are described below. N-alkylation . N-alkylation was performed as described previously [9]. Typically, to 100 mg of H₂T-2(3)-PyP (Frontier Scientific) in 20 mL DMF at 100°C was added 4 mL of the corresponding p-toluenesulfonate. Reaction was followed by thin-layer chromatography on silica gel TLC plates using 8:1:1 acetonitrile:KNO₃-saturated H₂O:H₂O as the mobile phase. Duration of N-alkylation is dependent upon the type of isomer and length of alkyl chains and ranged between few hours to few days. The course of N-alkylation was followed by TLC until no further change was observed. With meta isomers a single spot marked the completion of N-alkylation, while atropoisomers were observed with H₂TnHep-2-PyP. Upon completion, the reaction mixture was poured in a separatory funnel containing 200 ml each of water and chloroform and shaken well. The chloroform layer was discarded and extraction with CHCl₃ was repeated several times. The aqueous layer was filtered and the porphyrin was precipitated as PF₆⁻ salt by dropwise addition of a concentrated aqueous solution of NH₄PF₆, until no further precipitate formed. The precipitate was filtered and thoroughly washed with diethyl ether. After air drying the precipitate was dissolved in acetone, filtered and precipitated as the chloride salt by the addition of an acetone solution of tetrabutylammonium chloride. The precipitate was washed thoroughly with acetone and dried in vacuum at room temperature. Metallation. Metallation was achieved as previously described [9]. In brief, 20-fold excess MnCl₂ is added to a pH 12.5 solution of porphyrins at 25 °C. Metallation occurs in a few hours at room temperature for butyl, but requires overnight heating at 100°C for the heptyl and hexyl porphyrins. The reaction progress was followed by uv/vis spectroscopy and TLC (8:1:1=acetonitrile:KNO₃-saturated H₂O:H₂O) until the disappearance of both Soret band and fluorescence of the metal-free porphyrin. Upon completion, the suspension (excess of free Mn in the form of Mn oxo/hydroxo species) was filtered off, and the filtrate was precipitated with aqueous solution of NH₄PF₆ and washed with ether. The precipitate was dissolved in acetone, precipitated with an acetone solution of tetrabuthylammonium chloride and the resulting precipitate was washed with acetone. The PF₆⁻_(aq)/Cl⁻_(acetone) precipitation procedure was repeated twice to assure removal of free manganese.

Metal-free porphyrins and their metal complexes were characterized by thin-layer chromatography, elemental analysis, UV/vis spectroscopy and electrospray ionization mass spectrometry (ESI-MS). UV/vis data: the molar absorption coefficients and corresponding λ_max are summarized in Table 1, whereas ESI-MS data for the complexes are given in Table 2 and the peak assignments are consistent with those described for related compounds [30]. Elemental analysis: H₂TnBu-3-PyPCl₄·9H₂O. (C₅₆H₈₀N₈O₉Cl₄) Found: C, 58.99; H, 6.77; N, 9.88; Cl, 12.43 Calculated: C, 58.43; H, 7.00; N, 9.73; Cl, 12.31 %. H₂TnHex-3-PyPCl₄·13H₂O (C₆₄H₁₀₄N₈O₁₃Cl₄) Found: C, 57.49; H, 8.63; N, 8.49; Cl, 10.45 Calculated: C, 57.56; H, 7.85; N, 8.93; Cl, 10.45 %. H₂TnHep-2-PyPCl₄·12.5 H₂O (C₆₈H₁₁₁N₈O_12.5 Cl₄) Found: C, 58.93; H, 7.54; N, 8.12; Cl, 10.24 Calculated: C, 59.08; H, 8.08; N, 8.11; Cl, 10.26 %. MnTnBu-3-PyPCl₅·14H₂O (MnC₅₆H₈₈N₈O₁₄Cl₅) Found: C, 50.59; H, 6.76; N, 8.45; Cl, 13.45 Calculated: C, 50.74; H, 5.85; N, 8.42; Cl, 13.33 %. MnTnHex-3-PyPCl₅·13H₂O Found: C, 53.80; H, 6.47; N, 7.88; Cl, 12.33 Calculated: C, 53.99; H, 7.22; N, 7.87; Cl, 12.33 %. MnTnHep-2-PyPCl₅·20.5H₂O (MnC₆₈H₁₂₅N₈O_20.5Cl₅) Found: C, 50.43; H, 6.42; N, 7.24; Cl, 9.99 Calculated: C, 50.45; H, 8.02; N, 6.82; Cl, 10.85 %.

Table 1.

Molar absorption coefficients of chloride salts of metal-free porphyrins and their Mn(III) complexes synthesized in this work. The data for other compounds can be found elsewhere [9, 29]^[5] .

Porphyrin	λmax ( log εa)
H2TnBu-3-PyP	810.0(2.72); 638.5 (3.09); 582.0 (3.85); 514.5 (4.28); 417.5(5.53); 262.5 (4.41)
H2TnHex-3-PyP	830.0 (3.08); 823.0(3.05); 582.0 (3.76); 525.0 (4.20); 417.5 (5.43); 330.0 (4.28); 317.0 (4.25); 288.0 (4.08); 263.5 (4.34)
H2TnHep-2-PyP	639.0 (3.47); 584.0 (3.86); 543.0 (3.65); 510.5 (4.27); 416.5 (5.34); 263.0 (4.36)
MnTnBu-3-PyP	843.0 (2.54); 830.0 (2.46); 786.0 (3.27); 675.0 (3.43); 558.0 (4.04); 502.5 (3.75); 460.0 (5.06); 396.0 (4.60); 373.5 (4.62); 262.0 (4.50)
MnTnHex-3-PyP	565.5 (3.29); 675.5 (3.18); 557 (4.03); 501.5 (3.75); 460.0 (5.06); 395.5 (4.57); 372.0 (4.59); 330.5 (4.43); 262.0 (4.45)
MnTnHep-2-PyP	779.5 (3.23); 559.5 (4.08); 502.5 (3.79); 454.0 (5.19); 414.5 (4.45); 364.5 (4.66); 261.5 (4.49)

^aMolar absorption coefficient (M⁻¹ cm⁻¹) were determined in water at room temperature (± 5%). λ_max (nm) were determined with errors within 0.5 nm.

Table 2.

Electrospray ionization mass spectrometry of Mn(III) N-alkylpyridylporphyrins. Samples at 5 μM concentration were prepared in a H₂O-acetonitrile (1:1, v/v; containing 0.1 % v/v HFBA).

Peak Assignment	m/z
	MnTnBu-3-PyPCl5		MnTnHex-3-PyPCl5		MnTnHep-2-PyPCl5
	Found	Calculated	Found	Calculated	Found	Calculated
[MnT(alkyl)-2,3-PyP5+ + 2HFBA−]3+/3	442.3,	442.1	479.7	479.7	498.4	498.2
[MnT(alkyl)-2,3-PyP5+ + HFBA—2H+]2+/2	-	600.6	612.5	612.2	639.5	640.2
[MnT(alkyl)-2,3-PyP5+ + 2HFBA− - H+]2+/2	663.2,	662.6	-	718.7	746.8	746.7
[MnT(alkyl)-2,3-PyP5+ + 3HFBA−]2+/2	769.5	769.6	825.7	825.7	853.5	853.8

Thin-layer chromatography

Thin-layer chromatography of the purified compounds was performed on two types of silica gel TLC plates (plastic- or aluminum-backed) with 1:1:8 KNO₃-saturated H₂O: H₂O: acetonitrile mixture as mobile phase. Typically, 1 μL of ~1 mM samples were applied at ~ 1cm of the strip border and the solvent front was allowed to run ~12 cm. The absolute R_f values and their ratio thereoff are sensitive to the degree of the saturation of vapor phase in the TLC chamber and may differ slightly from one to another experiment. Thus, the internal standardization of TLC is required for comparison purposes [30].

Partition coefficient

Partition between water and n-butanol was determined experimentally with more lipophilic ortho and meta isomeric Mn(III) meso-tetrakis-N-alkylpyridylporphyrins using a variation of the shake-flask method [28]. Briefly, 0.1 mL of a MnP solution in n-butanol-saturated water was shaken with 1 mL of water-saturated n-butanol for 3 min using Vortex-Genie in plastic tube. The biphasic mixture was centrifuged (3 min at 6000 rpm) and the layers were separated. The concentration of the MnP in each layer was measured spectrophotometrically. If dilution was needed for the UV/vis measurements, the aqueous layer was diluted with water, whereas for the organic layer, water-saturated n-butanol was used instead. Spectrum baseline was chosen accordingly. The log P_BW was calculated using equation (1):

$$log{P}_{BW}=log\left(\frac{c_{\mathit{nBuOH}}}{c_{H2O}}\times \frac{V_{\mathit{nBuOH}}}{V_{H_{2}O}}\right)$$ (1)

The log P_BW values were converted to log P_OW using equation (2) as established recently [28] employing compounds that can be easily measured in both n-octanol and n-butanol (caffeine, pyridine, cyclohexanone, pyridazine, thiourea).

$$log{\text{P}}_{\text{OW}}=1.55·(log{\text{P}}_{\text{BW}})-0.54$$ (2)

The P_OW values for the hydrophilic members of the porphyrin series were calculated from the log P_OW vs R_f correlation defined in this work (see below).

SOD-like activity

Determination of the catalytic rate constant for O₂^·− dismutation using cyt c assay was performed as previously described in details [9].

Results

SOD-activity

All MnP studied here are good-to-potent SOD mimics. The catalytic rate constants for the dismutation of O₂^·− are given in Table 3.

Table 3.

The characterization of the lipophilicity and SOD-like activity of chloride salts of ortho and meta isomers of Mn(III) N-alkylpyridylporphyrins. R_f values refer to those calculated using plastic-backed TLC plates (R_f(Plastic)).

Porphyrin	Rf	log POW (determined)a	log POW (predicted)	log kcat
MnTM-2-PyP	0.03		−7.40c	7.79
MnTE-2-PyP	0.06		−6.43c	7.76
MnTnPr-2-PyP	0.11		−5.47c	7.38
MnTnBut-2-PyP	0.19	−4.64		7.25
MnTnHex-2-PyP	0.38	−2.29		7.48
MnTnHep-2-PyP	0.46	−1.63		7.65
MnTnOct-2-PyP	0.49	−0.77		7.71
MnTM-3-PyP	0.05		−6.49d	6.61
MnTE-3-PyP	0.10		−5.41d	6.83
MnTnPr-3-PyP	0.22	−4.53		6.85
MnTnBut-3-PyP	0.40	−3.56		6.74
MnTnHex-3-PyP	0.55	−1.60		6.72

^alog P_BW was determined experimentally using equation (1) and was converted to P_OW using equation (2);

^bconditions: 0.05 M phosphate buffer, 0.1 mM EDTA, pH 7.8, 40 μM xanthine, ~ 2nM xanthine oxidase, 10 μM cytochrome c. The rate of cytchrome c reduction by O₂^·−, k_{cyt c} = 2.6 x 10⁵ M⁻¹ cm⁻¹ is used for calculation of k_cat [44]. The estimated errors are within ±10 %.

^cCalculated according to eq. (5).

^dCalculated according to eq. (6).

Thin-layer chromatography

The R_f values measured for the compounds in silica gel plates (plastic-backed, R_f(Plastic)) are given in Table 3. A linear relationship between R_f(Plastic) values and the corresponding values obtained using aluminum-backed plates (R_f(Al)) was obtained and is depicted in Figure 2. Our data show that both plates exhibit equally well the effect of the lengthening of the alkyl chains. Because of such a linear relationship and for convenience, all R_f vales from hereon will refer to those calculated using plastic-backed TLC plates (R_f(Plastic)).

Figure 2.

The relationship between R_f obtained on TLC plastic-backed silica gel plates (R_f(Plastic)) and on TLC aluminum-backed silica gel plates (R_f(Al)) in acetonitrile: KNO₃-saturated H₂O:H₂O = 8:1:1. The ortho porphyrins are presented as squares (from methyl to octyl) and meta as triangles (from methyl to hexyl). The relationship is described with equation R_f(plastic) = 0.91 x R_f(Al) + 0.01, R² = 0.9609.

Partition coefficients

The log P_OW for the MnPs are given in Table 3. Linear relationships between log P_OW and the number of carbon atoms in the alkyl chain were observed in the series of both isomers (Fig. 3). Of note, all meta isomers (eq. 3) are more lipophilic than their respective ortho analogues (eq. 4).

Figure 3.

Relationship between the log P_OW and the number of carbon atoms (nC) for the series of ortho and meta Mn(III) N-alkylpyridylporphyrins. Full squares (ortho isomers) and triangles (meta isomers) present experimental values (determined in water/n-butanol system and converted to water/n-octanol system using equation 2) while empty squares and triangles present predicted values, respectively. The porphyrins are from bottom to top: MnTM-2,3-PyP, MnTE-2,3-PyP, MnTPr-2,3-PyP, MnTnBu-2,3-PyP, MnTnHex-2,3-PyP, MnTnHep-2-PyP, MnTnOct-2,3-PyP.

$$log{{\text{P}}_{\text{OW}}}^{(\mathit{ortho}\text{series})}=0.97·\text{nC}-8.36$$ (3)

$$log{{\text{P}}_{\text{OW}}}^{(\mathit{meta}\text{series})}=0.98·\text{nC}-7.46$$ (4)

Linear correlations between the log P_OW and the R_f values were also observed (Figure 4, eqs. 5 and 6). This provides an easy tool for prediction of log P_OW values of hydrophilic Mn porphyrins, of which direct measurement of log P_OW values is experimentally inaccessible. Such an approach has been used for calculating the log P_OW of the lower members of the ortho and meta series, including MnTE-2-PyP, as presented in Table 3.

Figure 4.

Relationship between the thin-chromatographic parameter, R_f and log P_OW for the series of ortho and meta Mn(III) N-alkylpyridylporphyrins. R_f values refer to those calculated using plastic-backed TLC plates (R_f(Plastic)). Full squares (ortho isomers) and triangles (meta isomers) present experimental values (determined in n-butanol/water system and converted to n-octanol/water system using equation 2) while empty squares and triangles present predicted values, respectively. The porphyrins are from bottom to top: MnTM-2,3-PyP (2,3-Me), MnTE-2,3-PyP (2,3-Et), MnTPr-2,3-PyP (2,3-Pr), MnTnBu-2,3-PyP (2,3-Bu), MnTnHex-2,3-PyP (2,3-Hex), MnTnHep-2,3-PyP (2,3-Hep), MnTnOct-2,3-PyP (R_f value was determined on silica gel plates with plastic base in acetonitrile: KNO₃-saturated H₂O:H₂O = 8:1:1.) Equations for ortho series is log P_OW = 12.69 x R_f - 7.59 (R² = 0.9672) and for meta series is log P_OW = 8.89 x R_f - 6.70 (R² = 0.9316).

$$log{{\text{P}}_{\text{OW}}}^{(\mathit{ortho}\text{series})}=12.69·{\text{R}}_{\text{f}}-7.59$$ (5)

$$log{{\text{P}}_{\text{OW}}}^{(\mathit{meta}\text{series})}=8.89·{\text{R}}_{\text{f}}-6.70$$ (6)

Discussion

While development of MnP-based experimental therapeutics of tailored bioavailability have been intensively pursued [26, 27], limitations of analytical nature have often precluded appropriate quantification of the lipophilicity of the candidate drugs [31]. In the particular case of Mn(III) ortho N-alkylpyridylporphyrins, this issue has been alternatively worked around by describing the lipohilic character of the therapeutics by using their chromatographic R_f values. Such characterization of lipophilicity, while sound and relevant to chemists, is less descriptive and meaningful from a pharmaceutical/medical point of view. Partition coefficient between n-octanol and water is a common pharmaceutical measure of drug lipophilicity/bioavailability. Therefore, for a broader audience, log P_OW values would relate more convincingly the increase in the in vivo efficacy of a drug to its lipophilicity, and would also allow comparison of MnPs to other drugs of similar therapeutic use. A strategy to overcome the technical difficulties in determining P_OW values of compounds that do not distribute directly into the n-octanol phase at levels measurable by convenient methods (e.g., cationic MnP) may involve the use of an alternative solvent to n-octanol (such as n-butanol) and a corresponding calibration curve relating P_OW to the partition in the new solvent [32]. This approach was recently shown adaptable to porphyrin P_OW measurements [28] and was used here. The n-butanol/water distribution of the more lipophilic cationic Mn porphyrins was then measured and converted to n-octanol/water partition (eq. 2, Table 3). The P_OW for the hydrophilic members of the porphyrin series were calculated from the appropriate log P_OW vs R_f relationship (Table 3).

The log P_OW vs nC

Our data show that the log P_OW is linearly related to the number of carbon atoms in the alkyl side chain (nC) for both ortho and meta isomers. The slope of the curves is close to one in both systems, which means that each CH₂ group in the side chain contributes to an ~10-fold increase in lipophilicity. Additionally, the parallel displacement of the meta isomer series by 1 log unit relative to the ortho series indicates that the meta isomers are ~10-fold more lipophilic than the corresponding ortho species (Figs. 3 and 4). Overall, this means that meta compounds share the same lipophificity of the ortho isomers that bear alkyl chains with one more CH₂ group. Thus, the same lipophilicity of a shorter meta hexyl species (MnTnHex-2-PyP) was achieved with the longer ortho heptyl porphyrin, MnTnHep-2-PyP (Table 3).

The log P_OW vs R_f

We established that with both meta and ortho isomers the log P_OW values are linearly related to R_f values (Figure 3). It follows, thus, that both parameters are equally valid means for describing MnP lipophilicity. The linear correlation also indicates that significant differences in lipophilicity correspond with small differences in R_f values; e.g., R_f changes of 0.1 for the ortho isomers result in an ~17-fold lipophilicity increase (1.24 log P_OW change) (Table 3). The 0.32 difference in R_f values between ortho ethyl and hexyl porphyrins (MnTE-2-PyP and MnTnHex-2-PyP) yields 4 orders of magnitude difference in P_OW value.

As opposed to the log P_OW vs nC relationships, the log P_OW vs R_f plots of the ortho and meta series are not parallel to each other. The spatial (ortho vs meta) substitution pattern controls the overall exposure of the porphyrin core and, hence, the adsorption of the compounds onto silica gel [33]. As the core exposure in the meta isomers is considerably less affected by the length of the alkyl side-chain (nC) than the ortho analogues, the dependence of R_f values on nC are less pronounced in the meta isomers. Thus, the log P_OW are more responsive to R_f values in the ortho series than in the meta series, as indicated by the greater slope of eq. 5 compared to that of eq. 6).

Therapeutics that are widely used in clinics have log P values in a range from ca −3 to ca + 5 [34 – 36]. The members of the ortho series with higher lipophilicity (P_OW values ≥ ca −2) and in vivo efficacy, such as MnTnOct-2-PyP, MnTnHep-2-PyP, MnTnHex-2-PyP) (Table 2), may be the most promising Mn porphyrins for clinical development, particularly for CNS injuries. In five in vivo and in vitro models of oxidative stress injury MnTnHex-2-PyP proved up to 120-fold more effective than MnTE-2-PyP, offering protection at submicromolar concentrations in cell models and at only 0.05 mg/kg in animal models. In two studies (cerebral palsy and ataxia telangiectasia) the hydrophilic MnTE-2-PyP was ineffective. The models that employed MnTnHex-2-PyP are: E. coli [27], ataxia telangiectasia [37], kidney ischemia/reperfusion [38], chronic morphine tolerance [26], lung radioprotection ¹ , stroke ², cerebral palsy ³, amyotrophic lateral sclerosis G93A mouse model ⁴. In a hippocampus organotypic slice model the octyl analogue, MnTnOct-2-PyP, was 3000-fold more protective against oxygen and glucose deprivation than the hydrophilic MnTE-2-PyP [43].

Ortho vs meta isomeric Mn(III) N-alkylpyridylporphyrins

We have previously observed that with ortho isomers the SOD activity (k_cat) drops from methyl to butyl and then increases again from butyl to octyl porphyrin [9] The data were discussed with respect to the interplay of electrostatic/steric and thermodynamic factors. However, there is essentially no difference in k_cat among meta isomers (Table 3), which is consistent with the special charge distribution/eletrostatics experienced by the Mn center in the meta series [40]. Consequently, as the alkyl chain grows from methyl to butyl the difference in k_cat between ortho and meta analogues drops from 15-fold for methyl to 3.2-fold for butyl porphyrin (Table 2), while the difference in lipophilicity remains the same (Figure 4). Further, meta isomers are less bulky. We have shown with methyl analogues that meta isomer, MnTM-3-PyP accumulates more in E. coli than bulkier ortho porphyrin MnTM-2-PyP [41]; the protection of aerobic growth of SOD-deficient E. coli with ortho and meta isomers was nearly identical. The unfavorable impact of bulkiness is much more pronounced with bigger molecules [27, 41, 42]. Although imidazolium derivatives (MnTDE-2-ImP and MnTDMOE-2-ImP) [27, 43] are equally potent SOD mimics, they accumulate less in E. coli and are therefore less protective than MnTE-2-PyP [27]. In summary, due to their higher lipophilicity and lesser bulkiness, meta porphyrins may accumulate to higher levels within a cell and that may compensate for somewhat lower k_cat; in turn they may be in vivo of similar efficacy as are ortho isomers (work in progress) ⁵.

According to Hansch et al [34] the ideal compound would have log P_OW = ~ 2; ortho Mn(III) tetrakis(N-undecylpyridinium-2-yl)porphyrin with log P_OW = 1.98 or its meta decyl analogue would meet such criteria. Yet, although of appropriate lipophilicity Mn(III) tetrakis(N-undecylpyridinium-2-yl)porphyrin is likely too big to easily accumulate within cell. As discussed above to predict the drug antioxidant efficiency in vivo, other parameters should be considered, particularly stericity. The size and the shape of the molecule would determine the bulkiness of the molecule, but would also affect the shielding of the positive charges; combined these effects would dominate the transport and the interactions (likely unfavorable) with biomolecules which MnP would encounters on its way through the cell. In addition to steric factors, the redox property, i.e. the reducibility of Mn⁺³ porphyrin site with cellular reductants, must also be accounted for while designing or choosing the appropriate MnP, as experimental therapeutics in oxidative stress models.

Conclusions

Partition of isomeric Mn(III) N-alkylpyridylporphyrins between n-octanol and water, P_OW, was determined. The log P_OW is linearly related to thin-layer chromatographic parameter, R_f and to the number of carbon atoms with both ortho and meta isomers. Thus both parameters accurately describe drug lipophilicity. Yet, log P_OW may be more convenient and meaningful for the pharmaceutical and medical community particularly for the comparison of MnP to other drugs of similar therapeutic target that are in clinics or under development. The longer ortho N-alkylpyridylporphyrins, such MnTnHex-2-PyP and MnTnOct-2-PyP are significantly more lipophilic than MnTE-2-PyP and thus may be promising therapeutics, particularly in the cases of central nervous system injuries. Our data also show that meta isomers are 10-fold more lipophilic than ortho porphyrins and are in addition less bulky. Favorable lipophilicity and stericity would compensate for the lower antioxidant potency making meta isomers prospective antioxidants in vivo also.

Abbreviations Charges are omitted throughout text

MnP

Mn porphyrin

MnTalkyl-2

3-PyP⁵⁺ Mn(III) meso-tetrakis(N-alkylyridinium-2 or 3-yl)porphyrin, alkyl being methyl (M, AEOL10112), ethyl (E, AEOL10113), n-propyl (nPr), n-butyl (nBu), n-hexyl (nHex), n-heptyl (nHep), n-octyl (nOct)

2 and 3 relate to ortho and meta isomers

respectively

MnTDE-2-ImP⁵⁺

Mn(III) tetrakis(N,N’-diethylimidazolium-2-yl)porphyrin, AEOL10150

MnTDMOE-2-ImP⁵⁺

Mn(III) tetrakis[N,N’-di(2-methoxethyl)imidazolium-2-yl)porphyrin

P_OW

partition coefficient between n-octanol and water

P_BW

partition coefficient between n-butanol and water

TLC

thin-layer chromatography

E_1/2

half-wave reduction potential

SOD

superoxide dismutase

HIF-1α

hypoxia inducible factor-1, NF-κB, nuclear factor κB

AP-1

activator protein-1