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A Nonpeptidyl Mimic of Superoxide Dismutase with Therapeutic Activity in Rats
Daniela Salvemini, Zhi-Qiang Wang, Jay L. Zweier, Alexandre Samouilov, Heather Macarthur, Thomas P. Misko, Mark G. Currie, Salvatore Cuzzocrea, James A. Sikorski, and Dennis P. Riley

Supplementary Material

M40403 Synthesis. The synthesis of M40403 is based on the utilization of the Mn(II) ion as a template for executing a highly disfavored cyclization reaction producing a 15-membered ring, pyridinediimine complex, in quantitative yield. This atom-efficient one-step assembly of the precursor diimine complex is followed by a routine NaBH4 reduction to afford the desired pure M40403 after crystallization from water. This complex and a related dimethyl derivative, M40404, which is catalytically inactive for the superoxide dismutase (SOD) reaction, are readily synthesized from commercially available pyridine precursors (pyridine-2,6-dicarboxaldehyde and 2,6-diacetylpyridine, respectively) and are prepared chemically and optically pure in high yields, exceeding 90%. The synthesis of M40403, an all R-bis-cyclohexano substituted macrocyclic ring complex is accomplished through this template ring closure based on the method of N. W. Alcock, D. C. Liles, M. McPartlin, P. A. Tasker, J. Chem. Soc. Chem. Commun. 1974, 727 (1974), in which the linear tetraammine, N,N´(-bis-{(1R,2R)-[2-(amino)]cyclohexyl}-1,2-diaminoethane, is used in place of the tetraammine, N,N´(-bis{2-(amino)phenyl} -1,2-diaminoethane. Details of the synthesis are in a manuscript submitted to Inorganic Chemistry. Mass spectrometry (LRFAB) mass-to-charge ratio (m/z) = 447 [M - Cl]+. Analysis calculated for C21H35Cl2N5Mn: C, 52.18; H, 7.30; N, 14.49; Cl, 14.67. Found: C, 51.89; H, 7.35; N, 14.26; Cl, 14.55. All values are given as percentages.

M40403 Chemical Stability. The chemical stability (kinetic) of the complex M40403 has been determined through methods described previously [D. P. Riley and R. H. Weiss, J. Am. Chem. Soc.116, 387 (1994); D. P. Riley et al., Inorg. Chem. 35, 5213 (1996); D. P. Riley, S. L. Henke, P. J. Lennon, R. H. Weiss, W. L. Neumann, J. Am. Chem. Soc. 119, 6522 (1997)] and possesses in water a second-order dependence (first-order in [H+] and first-order in [M40403]) for the rate of loss of ligand from the Mn(II) center. This rate constant is 0.135 M-1 s-1, which corresponds to a half-life at pH 5 for dissociation to free ligand of ~6 days. The thermodynamic binding (stability) constant is remarkably high for a Mn(II) complex and cannot be measured with accuracy because of the extremely slow kinetics of ligand dissociation, but a lower limit has been established as log K > 17. The complex is also resistant to oxidative degradation as its oxidation potential for the Mn(II)/(III) couple is +0.74 V (reversible, versus standard hydrogen electrode). The SOD catalytic rate constant is pH dependent with a rate at pH 6.0 and 21°C exceeding 2 108 M-1 s-1 or comparable to the native Mn SOD enzyme, whereas at pH =7.4, the rate is 2.0 10+7 M-1 s-1 and 21°C.

M40403 Plasma and in Vivo Stability. The in vivo and in vitro stability of M40403 was assessed with several techniques for monitoring the intact complex in biological samples. These included (i) standard reverse-phase high-performance liquid chromatography (HPLC) with a 250 ( 4.6 mm, 5 μm, C18 Vydac (Hesperia, CA) 218TP54 protein and peptide column at 25°C for monitoring the stability of the complex in buffer and in rat plasma and (ii) the uniqueness of the electron paramagnetic resonance (EPR) spectra of M40403, which makes it possible to detect its presence quantitatively with EPR regardless of its environment (biological matrix). In the frozen state at 77 K, the intact complex produces a broad EPR spectrum over 5000 G with four distinct peaks, whereas free Mn(II) produces a much sharper spectrum of six well-resolved peaks (equal intensity) centered near EM>g = 2 (Fig. 1). At room temperature, the complex is not detectable, whereas free Mn(II) produces the spectrum with six sharp peaks. Spectra were recorded at 77°C on a Bruker ESP 300 spectrometer [J. L. Zweier, R. Brodericks, P. Kuppusamy, S. Thompson-Gorman, G. A. Lutty, J. Biol. Chem.269, 24156 (1994)]. This spectrum of free Mn(II) allows one to quantitate the release of free Mn(II) from the complex. The release profile in rat blood was monitored for M40403 through incubation at 37°C, the spectra were fitted with the known Mn(II) spectrum, and concentrations were calculated with the linear calibration curve for the control Mn(II) EPR spectra . For up to 6 hours (duration of monitoring), no release of free Mn(II) was observed in the whole blood. Partitioning in blood was estimated by fitting EPR spectra of blood fractions with the spectra of the Mn(II) complex (M40403). Blood was spun down after incubation with drug (1.12 mM) at 37°C. Analysis of data shows that the drug has a tendency to be concentrated in the spun-down cell fraction. We also determined the relative stability of M40403 in rat plasma at 37°C by HPLC, which enables quantitation of the complex with a sensitivity of ~75 μM. No change in the concentration of the complex was observed in buffer or plasma after 6 hours at 37°C. Comparison of the initial peak areas between the plasma sample and buffer control showed <1% deviation, evidence that little or no complex is adsorbed to or coelutes with plasma constituents. Observation of M40403 in incubated whole blood showed considerable difference to the plasma study. After only 30 min, the [M40403] remaining in the liquid phase after spinning down the cells declined ~60%. The material that remains in the extracellular phase (centrate) is completely stable over the period of monitoring (10 hours). These results indicate that M40403 is taken up by red blood cells. The application of the EPR method allowed us to directly quantitate the amount of M40403 associated with the cellular fraction of the blood and to show that the complex is completely intact in this fraction. M40403 in blood showed no free Mn(II) after 6 hours of incubation when examined with EPR or HPLC. Interestingly, the compound partitioned into the cellular fraction of the blood as noted from the HPLC assay. The complex remained intact over the course of the experiment in both the cellular and plasma fractions as monitored by both HPLC and EPR methods.

In Vivo Distribution of M40403. Partitioning of the drug in a whole animal was accomplished by intravenous (iv) injection of 1.12 (mol of M40403 into rats and monitoring through EPR the distribution of the drug for 10 hours after killing the animal. The distinctive spectrum of the complex was fitted and integrated to afford a quantitative readout of the distribution of the drug in vivo. Using the inherent greater sensitivity provided by in vivo EPR detection, we monitored the biodistribution of the complex. After an iv injection of 1.12 μmol of the drug into rats, animals were killed after 6 hours, and blood, urine, and organ samples were collected and subjected to the EPR analysis for intact complex. In all cases, no free Mn(II) was detected. The quantity (concentration) of drug observed in the urine was 37 μM, and the quantity in the liver approached 3 μM. Additionally, 2.4, 0.7, 0.6, 0.35, 0.5, and 0.2 μM was observed in the kidney, lungs, spleen, brain, heart, and blood, respectively. The complex was widely distributed in the whole animal but, nevertheless, tended to remain associated with more hydrophilic sites.

Synthesis of M40404. The all R-substituted complex M40404 was prepared in an analogous manner to M40403, but 2,6-diacetylpyridine was used in place of the 2,6-pyridine dicarboxaldehyde. The diimine precursor complex was reduced with sodium borohydride to give with complete stereoselectivity the SOD-inactive all R-bis-dimethyl substituted derivative M40404. MS (LRFAB) m/z 475 [M - Cl]+. Analysis calculated for C23H39Cl2N5Mn: C, 54.01; H, 7.69; Cl, 13.86; N, 13.69. Found: C, 53.65; H, 7.61; Cl, 13.11; N, 12.90. All values are given as percentages.

Fig. 1. EPR spectra of M40403 and free Mn(II). EPR spectra were measured on 1.12 mM solution in Hepes buffer at pH 7.4 at (left) 77 K or at (right) 297 K with a Bruker ER 300 spectrometer

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Fig. 2. The decrease in mean arterial blood pressure in splanchnic artery occlusion rat models (control) is blocked by M40403 (1 mg/kg) but not by the inactive SOD mimic, M40404 (1mg/kg). Sham animals are also shown. Each point is the mean ± SEM (error bars) for eight experiments.

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Supplemental Table 1. The intraplantar injection of carrageenan elicits a time-dependent release of prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) in the paw exudates. M40404 had no effect (10 mg/kg; given as iv bolus). Each point is the mean ± SEM for six experiments. MPO, myeloperoxidase; LDH, lactate dehydrogenase.
Time after carrageenan injection (hours) MPO (mU per paw) TNF-α (pg per paw) IL-1β (pg per paw) Time after carrageenan injection (hours) LDH (U per paw) PGE2 (ng per paw)
0 214 ± 12 0 0 0 25 ± 7 0.4 ± 0.1
1 4596 ± 93 500 ± 10 200 ± 30 1 50 ± 3 0.74 ± 0.03
3 3505 ± 367 820 ± 15 900 ± 40 3 325 ± 26 3 ± 0.1
6 3636 ± 51 950 ± 35 800 ± 30 6 300 ± 20 3 ± 0.2
6 + M40404 4000 ± 71 840 ± 50 825 ± 25 6 + M40404 310 ± 5 4 ± 1
Supplemental Table 2. Values represent levels of mediators in blood and tissue at various stages of the SAO model. Increased levels of these mediators are seen only after the period of reperfusion (after reperfusion). Ischemia alone does not cause inflammation (values before ischemia versus values after ischemia).
Mediator Before ischemia After reperfusion After ischemia
Plasma malondialdehyde 23 ± 2 64 ± 6 17 ± 5
TNF-α 0 ± 0 205 ± 77 0 ± 0
IL-1β 0 ± 0 139 ± 5 0 ± 0
Ileum MPO 100 ± 10 400 ± 15 90 ± 10
Lung MPO 1142 ± 128 2589 ± 414 1000 ± 100