U.S. patent application number 12/025612 was filed with the patent office on 2008-08-28 for substituted porphyrins.
This patent application is currently assigned to Duke University. Invention is credited to Ines Batinic-Haberle, Irwin Fridovich, Ivan Spasojevic.
Application Number | 20080207582 12/025612 |
Document ID | / |
Family ID | 29736167 |
Filed Date | 2008-08-28 |
United States Patent
Application |
20080207582 |
Kind Code |
A1 |
Batinic-Haberle; Ines ; et
al. |
August 28, 2008 |
Substituted Porphyrins
Abstract
A series of ortho isomers of meso tetrakis
N-alkylpyridylporphyrins (alkyl being methyl, ethyl, n-propyl,
n-butyl, n-hexyl, and n-octyl) and their Mn(III) complexes were
synthesized and characterized by elemental analysis, uv/vis
spectroscopy, electrospray ionization mass spectrometry and
electrochemistry. An increase in the number of carbon atoms in the
alkyl chains from 1 to 8 is accompanied by an increase in: (a)
lipophilicity measured by the chromatographic retention factor,
R.sub.f; (b) metal-centered redox potential, E.sub.1/2 from +220 to
+367 mV vs NHE, and (c) proton dissociation constant, pK.sub.a2
from 10.9 to 13.2. A linear correlation was found between E.sub.1/2
and R.sub.f of the Mn(III) porphyrins and between the pK.sub.a2 and
R.sub.f of the metal-free compounds. As the porphyrins become
increasingly more lipophilic, the decrease in hydration disfavors
the separation of charges, while enhancing the electron-withdrawing
effect of the positively charged pyridyl nitrogen atoms.
Consequently, the E.sub.1/2 increases linearly with the increase in
pK.sub.a2, a trend in porphyrin basicity opposite from the one we
previously reported for other water-soluble Mn(III) porphyrins. All
of these Mn(III) porphyrins are potent catalysts for superoxide
dismutation (disproportionation). Despite the favorable increase of
E.sub.1/2 with the increase in chain length, the catalytic rate
constant decreases from methyl (log k.sub.cat=7.79) to n-butyl, and
then increases such that the n-octyl is as potent an SOD mimic as
are the methyl and ethyl compounds. The observed behavior
originates from an interplay of hydration and steric effects that
modulate electronic effects.
Inventors: |
Batinic-Haberle; Ines;
(Durham, NC) ; Spasojevic; Ivan; (Durham, NC)
; Fridovich; Irwin; (Durham, NC) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER, EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
Duke University
Durham
NC
|
Family ID: |
29736167 |
Appl. No.: |
12/025612 |
Filed: |
February 4, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10456956 |
Jun 9, 2003 |
|
|
|
12025612 |
|
|
|
|
60386454 |
Jun 7, 2002 |
|
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Current U.S.
Class: |
514/185 |
Current CPC
Class: |
C07D 487/22 20130101;
A61P 39/06 20180101; A61P 11/06 20180101; A61P 11/00 20180101 |
Class at
Publication: |
514/185 |
International
Class: |
A61K 31/555 20060101
A61K031/555 |
Claims
1.-5. (canceled)
6. A method of protecting cells from oxidant-induced toxicity
comprising contacting said cells with a protective amount of a
compound under conditions such that the protection is effected,
said compound having the formula ##STR00005## wherein each R is,
independently, an alkyl group of greater than 8 carbons, each A is,
independently, hydrogen or a halogen, M is a metal selected from
the group consisting of manganese, iron, copper, cobalt, nickel and
zinc, and Z.sup.- is a counterion.
7. The method according to claim 6 wherein said cells are mammalian
cells.
8. A method of treating a pathological condition of a patient
resulting from oxidant-induced toxicity comprising administering to
said patient an effective amount of a compound under conditions
such that the treatment is effected, said compound having the
formula ##STR00006## wherein each R is, independently, an alkyl
group of greater than 8 carbons, each A is, independently, hydrogen
or a halogen, M is a metal selected from the group consisting of
manganese, iron, copper, cobalt, nickel and zinc, and Z.sup.- is a
counterion.
9. A method of treating a pathological condition of a patient
resulting from degradation of NO.sup..cndot., comprising
administering to said patient an effective amount of a compound
under conditions such that the treatment is effected, said compound
having the formula ##STR00007## wherein each R is, independently,
an alkyl group of greater than 8 carbons, each A is, independently,
hydrogen or a halogen, M is a metal selected from the group
consisting of manganese, iron, copper, cobalt, nickel and zinc, and
Z.sup.- is a counterion.
10. A method of treating a patient for inflammatory lung disease
comprising administering to said patient an effective amount of a
compound under conditions such that the treatment is effected, said
compound having the formula ##STR00008## wherein each R is,
independently, an alkyl group of greater than 8 carbons, each A is,
independently, hydrogen or a halogen, M is a metal selected from
the group consisting of manganese, iron, copper, cobalt, nickel and
zinc, and Z.sup.- is a counterion.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a divisional of patent application Ser.
No. 10/456,956, filed Jun. 9, 2003 which claims priority from
Provisional Application No. 60/386,454, filed Jun. 7, 2002, the
content of which is incorporated herein by reference.
INTRODUCTION
[0002] Low-molecular weight catalytic scavengers of reactive oxygen
and nitrogen species, aimed at treating oxidative stress injuries,
have been actively sought. Three major groups of manganese
complexes have been developed and tested in vitro and in vivo; Mn
porphyrins,.sup.1-9 Mn cyclic polyamines.sup.10 and Mn salen
derivatives..sup.11 Based on a structure-activity relationships
that we developed for water-soluble Mn(III) and Fe(III)
porphyrins,.sup.2-4 Mn(III) meso
tetrakis(N-methylpyridinium-2-yl)porphyrin
(Mn.sup.IIITM-2-PyP.sup.5+, AEOL-10112) and meso
tetrakis(N-ethylpyridinium-2-yl)porphyrins
(Mn.sup.IIITE-2-PyP.sup.5+, AEOL-11013) were proposed and then
shown to be potent catalysts for superoxide dismutation..sup.4,12
The alkyl substitutions at the ortho positions restrict the
rotation of the pyridyl rings with respect to the porphyrin plane.
Consequently both compounds exist as mixtures of four
atropoisomers, all of which were shown to be equally potent
catalysts for O.sub.2.sup..cndot.- dismutation..sup.13 These Mn
porphyrins also allow SOD-deficient Escherichia coli to grow under
aerobic conditions,.sup.4,12 and offer protection in rodent models
of oxidative stress such as stroke,.sup.14 diabetes,.sup.15 sickle
cell disease,.sup.16 and cancer/radiation..sup.17 The high formal
+5 charge of these metalloporphyrins could influence their tissue
distribution, transport across biological membranes, and binding to
other biomolecules and their low lipophilicities may restrict their
protective effects. With the aim of modulating metalloporphyrin
subcellular distribution, higher N-alkylpyridylporphyrin analogues
(Scheme I) with increased lipophilicity were synthesized. We
anticipate that their comparative kinetic and thermodynamic
characterization will deepen our insight into the modes of action
of porphyrin-based catalytic antioxidants and the mechanisms of
oxidative stress injuries.
BRIEF DESCRIPTION OF THE DRAWINGS
[0003] FIG. 1. Structures of the most hydrophilic
(Mn.sup.IIITM2-PyP.sup.5+) and the most lipophilic
(Mn.sup.IIITnOct2-PyP.sup.5+) members of the series studied. The
.alpha..beta..alpha..beta. atropoisomers are shown.
[0004] FIG. 2. The lipophilicity, R.sub.f of
H.sub.2T(alkyl)-2-PyP.sup.4+ (A) and
Mn.sup.IIIT(alkyl)-2-PyP.sup.5+ compounds (B) vs the number of
CH.sub.2 groups.
[0005] FIG. 3. Proton dissociation constants pK.sub.a2 of the
metal-free porphyrins, H.sub.2T(alkyl)-2-PyP.sup.4+ (A), and the
metal-centered redox potentials E.sub.1/2 for the Mn(III)/Mn(II)
couple of Mn.sup.IIIT(alkyl)-2-PyP.sup.5+ porphyrins (B) as a
function of R.sub.f. Inserts: pK.sub.a2 (FIG. 3A) and E.sub.1/2
(FIG. 3B) vs the number of CH.sub.2 groups.
[0006] FIG. 4. The reactivity of water-soluble Mn(III) porphyrins
(A) (ref 4) and Mn.sup.IIIT(alkyl)-2-PyP.sup.5+ porphyrins (B) as
catalysts for O.sub.2.sup.- dismutation, expressed in terms of log
k.sub.cat vs E.sub.1/2.
[0007] FIG. 5. E.sub.1/2 for the Mn(III)/Mn(II) couple of
Mn.sup.IIIT(alkyl)-2-PyP.sup.5+ porphyrins vs pK.sub.a2 of the
corresponding metal-free ligands. Insert: E.sub.1/2 of
water-soluble Mn(III) porphyrins vs pK.sub.a3 (data from ref 4);
Mn.sup.IIITE-2-PyP.sup.5+ (1), MnTM-2-PyP.sup.5+ (2),
MnPTrM-2-PyP.sup.4+ (3), Mn.sup.IIITM-4-PyP.sup.5+ (4),
Mn.sup.IIITM-3-PyP.sup.5+ (5),
Mn.sup.IIIT(2,6-Cl.sub.2-3-SO.sub.3--P)P.sup.-3 (6),
Mn.sup.IIIT(TFTMA)P.sup.5+ (7),
Mn.sup.IIIT(.alpha..alpha..alpha..alpha.-2-MINP)P.sup.5+ (8),
Mn.sup.IIIT(2,6-Cl.sub.2-3-SO.sub.3--P)P.sup.3- (9),
Mn.sup.IIIT(2,4,6-Me.sub.3-3,5-(SO.sub.3).sub.2--P)P.sup.7- (10),
Mn.sup.III(TMA)P.sup.5+ (11), MnTSPP.sup.3- (12), MnTCPP.sup.3-
(13), Mn.sup.IIIhematoP.sup.- (14).
[0008] FIG. 6. Cyclic voltammetry of 0.5 mM
Mn.sup.IIITE-2-PyP.sup.5+ and Mn.sup.IIITnHex-2-PyP.sup.5+
porphyrins in a 0.05 M phosphate buffer (pH 7.8, 0.1 M NaCl) at a
scan rate of 0.1 V/s.
[0009] FIG. 7. Electrospray mass spectrometry of 0.5 mM solutions
of H.sub.2T(alkyl)-2-PyP.sup.4+ compounds in 1:1 water:acetonitrile
at a cone voltage of 20 V.
[0010] FIG. 8. Electrospray mass spectrometry of 0.5 mM solutions
of Mn.sup.IIIT(alkyl)-2-PyP.sup.5+ compounds in 1:1
water:acetonitrile at a cone voltage of 20 V.
[0011] FIG. 9. As the alkyl chains of Mn(III) porphyrins lengthen,
the favorable increase in E.sub.1/2 overcomes the unfavorable
steri/electrostatic effects such that the n-octyl is as potent a
catalyst of O.sub.2-- dismutation as are the methyl and ethyl
compounds.
DETAILED DESCRIPTION OF THE INVENTION
[0012] The present invention relates to a compound of formula
##STR00001## ##STR00002##
[0013] wherein
[0014] each R is, independently, an C1-C12 alkyl (preferably, a C8
to C12 alkyl),
[0015] each A is, independently, hydrogen or an electron
withdrawing group,
[0016] M is a metal selected from the group consisting of
manganese, iron, copper, cobalt, nickel and zinc, and
[0017] Z.sup.- is a counterion. In one embodiment, at least one A
is a halogen.
[0018] The invention further relates to a method of protecting
cells (eg mammalian cells) from oxidant-induced toxicity comprising
contacting the cells with a protective amount of a compound as
described above. The invention further relates to a method of
treating a pathological condition of a patient resulting from
oxidant-induced toxicity comprising administering to the patient an
effective amount of such a compound. The invention also relates to
a method of treating a pathological condition of a patient
resulting from degradation of NO.sup..cndot., comprising
administering to the patient an effective amount of a compound as
described above. Additionally, the invention relates to a method of
treating a patient for inflammatory lung disease comprising
administering to the patient an effective amount of a compound as
described above. The inflammatory lung disease can be a
hyper-reactive airway disease. The disease can be asthma.
[0019] The entire content of all documents cited herein are
incorporated herein by reference. Also incorporated herein by
reference is Batinic-Haberle et al, J. Chem. Soc., Dalton Trans.
2002, 2689-2696.
[0020] Also incorporated by reference is U.S. application Ser. No.
09/880,125, filed Jun. 14, 2001.
EXAMPLE
Experimental
Materials and Methods
[0021] General. MnCl.sub.2.times.4H.sub.2O, and Baker-flex silica
gel IB TLC plates were purchased from J. T. Baker.
N,N-dimethylformamide, ethyl p-toluenesulfonate, 2-propanol
(99.5+%), NH.sub.4 PF.sub.6 (99.99%), NaCl, sodium L-ascorbate
(99+%) and tetrabutylammonium chloride were from Aldrich, while
xanthine, and ferricytochrome c were from Sigma. The n-propyl,
n-butyl, n-hexyl and n-octyl esters of p-toluenesulfonic acid were
from TCI America. Methanol (anhydrous, absolute), ethanol
(absolute), acetone, ethyl ether (anhydrous), chloroform, EDTA and
KNO.sub.3 were from Mallinckrodt and acetonitrile was from Fisher
Scientific. Xanthine oxidase was prepared by R. Wiley and was
supplied by K. V. Rajagopalan..sup.18 Catalase was from Boehringer,
ultrapure argon from National Welders Supply Co., and tris buffer
(ultrapure) was from ICN Biomedicals, Inc.
[0022] H.sub.2T(alkyl)-2-PyP.sup.4+. Tetrakis(2-pyridyl)porphyrin,
H.sub.2T-2-PyP was purchased from Mid-Century Chemicals, Chicago,
Ill. The increased lipophilicity of the n-propyl, n-butyl, n-hexyl,
and n-octyl analogues required a slight modification of the
synthetic approach used for methyl and ethyl compounds..sup.4,12
Typically, 100 mg of H.sub.2T-2-PyP was dissolved in 20 mL of DMF
at 100.degree. C., followed by the addition of 4 mL of the
corresponding p-toluenesulfonate. The course of N-alkylation was
followed by thin-layer chromatography on silica gel TLC plates
using 1:1:8 KNO.sub.3-saturated H.sub.2O:H.sub.2O:acetonitrile as a
mobile phase. While complete N-alkylation is achieved within a few
hours for the methyl analogue, the required time gradually
increases and it took three and five days to prepare the n-hexyl
and n-octyl compounds, respectively. Upon completion, for the
methyl, ethyl and n-propyl compounds, the reaction mixture was
poured into a separatory funnel containing 200 mL each of water and
chloroform and shaken well. The chloroform layer was discarded and
the extraction with CHCl.sub.3 was repeated several times. The
n-butyl, n-hexyl and n-octyl analogues are more lipophilic and
tended to remain in the chloroform layer. Therefore, increasing
amounts of methanol were added to the water/CHCl.sub.3 mixture in
order to force the porphyrin into the aqueous/methanol layer. This
layer was filtered and the porphyrin was precipitated as the
PF.sub.6.sup.- salt by the addition of a concentrated aqueous
solution of NH.sub.4 PF.sub.6. The precipitate was thoroughly
washed with 1:1 2-propanol:diethylether in the case of methyl and
ethyl compounds and with pure diethylether for the others. The
precipitate was then dissolved in acetone, filtered and
precipitated as the chloride salt by the addition of
tetrabutylammonium chloride dissolved in acetone. The precipitate
was washed thoroughly with acetone, and dried in vacuo at room
temperature. Elemental analysis:
H.sub.2TnPr-2-PyPCl.sub.4.times.12.5H.sub.2O
(C.sub.52H.sub.71N.sub.8O.sub.12.5Cl.sub.4). Found: C, 54.2; H,
6.42; N, 9.91; Cl, 12.04. Calculated: C, 54.20; H, 6.18; N, 9.68;
Cl, 12.25. H.sub.2TnBut-2-PyPCl.sub.4.times.10.5
H.sub.2O(C.sub.56H.sub.75N.sub.8O.sub.10.5Cl.sub.4). Found: C,
57.16; H, 6.94; N, 9.513; Cl, 1.77. Calculated: C, 57.10; H, 6.41;
N, 9.51; Cl, 12.03. H.sub.2TnHex-2-PyPCl.sub.4.times.11 H.sub.2O
(C.sub.64H.sub.100N.sub.8O.sub.11Cl.sub.4). Found: C, 59.19; H,
7.31; N, 8.61; Cl, 11.09. Calculated: C, 59.16; H, 7.751; N, 8.60;
Cl, 10.91. H.sub.2TnOct-2-PyPCl.sub.4.times.13.5H.sub.2O
(C.sub.64H.sub.121N.sub.8O.sub.13.5Cl.sub.4). Found: C, 59.37; H,
7.41; N, 7.73. Calculated: C, 59.37; H, 8.37; N, 7.69.
[0023] Mn.sup.IIIT(alkyl)-2-PyP.sup.5+. Metalation of the
N-alkylated porphyrins was achieved as described previously for the
methyl and ethyl compounds..sup.4,12 Metal incorporation became
slower as the alkyl chains lengthened. Under same conditions
(20-fold excess metal, 25.degree. C., pH 12.3) it occurs almost
instantaneously for methyl and ethyl, within minutes for n-propyl,
in .about.30 minutes for n-butyl, in .about.1 hour with the
n-hexyl, and took several hours at 100.degree. C. for the n-octyl
porphyrin. The formation of the Mn(II) porphyrin and its oxidation
to Mn(III) were clearly distinguishable steps when the n-hexyl and
n-octyl analogues were metallated. As was the case with the
metal-free ligands, the PF.sub.6.sup.- salts of Mn(III) n-propyl,
n-butyl, n-hexyl and n-octyl compounds were washed only with
diethylether. Elemental analysis:
Mn.sup.IIITnPr-2-PyPCl.sub.5.times.11.5 H.sub.2O
(MnC.sub.52H.sub.75N.sub.8O.sub.11.5Cl.sub.5). Found: C, 50.90; H,
6.07; N, 9.27; Cl, 13.48. Calculated: C, 50.85; H, 6.16; N, 9.12;
Cl, 14.43. Mn TnBut-2-PyPCl.sub.5.times.12.5H.sub.2O
(MnC.sub.56H.sub.85N.sub.8O.sub.12.5Cl.sub.5). Found: C, 51.58; H,
6.33; N, 9.55; Cl, 15.53. Calculated: C, 51.64; H, 6.58; N, 8.60;
Cl, 13.61. Mn TnHex-2-PyPCl.sub.5.times.10.5H.sub.2O
(MnC.sub.64H.sub.97N.sub.8O.sub.12.5Cl.sub.5). Found: C, 55.64; H,
7.14; N, 8.23; Cl, 12.60. Calculated: C, 55.76; H, 7.09; N, 8.13;
Cl, 12.86. Mn.sup.IIITnOct-2-PyPCl.sub.5.times.10
H.sub.2O.times.2.5 NH.sub.4Cl
(MnC.sub.64H.sub.122N.sub.10.5O.sub.10Cl.sub.7.5). Found: C, 53.56;
H, 7.13; N, 9.12; Cl, 16.84. Calculated: C, 53.53; H, 7.60; N,
9.10; Cl, 16.46.
[0024] Thin-layer chromatography. All ligands and their Mn(III)
complexes were chromatographed on silica gel TLC plates using 1:1:8
KNO.sub.3-saturated H.sub.2O:H.sub.2O:acetonitrile. The
atropoisomers could not be separated for the methyl.sup.19 and
ethyl analogues,.sup.2-4 they begin to separate for the n-propyl
and n-butyl species and were clearly resolved with the n-hexyl and
n-octyl compounds.
[0025] Uv/vis spectroscopy. The uv/vis spectra were taken on a
Shimadzu UV-2501 PC spectrophotometer at 25.degree. C. The proton
dissociation constants (pK.sub.a2), were determined
spectrophotometrically at 25.degree. C., at an ionic strength of
0.1 M (NaOH/NaNO.sub.3), as previously described..sup.4
[0026] Electrochemistry. Measurements were performed on a CH
Instruments Model 600 Voltammetric Analyzer..sup.3,4 A
three-electrode system in a small volume cell (0.5 mL to 3 mL),
with a 3 mm-diameter glassy carbon button working electrode
(Bioanalytical Systems), plus the Ag/AgCl reference and Pt
auxillary electrodes was used. Solutions contained 0.05 M phosphate
buffer, pH 7.8, 0.1 M NaCl, and 0.5 mM metalloporphyrin. The scan
rates were 0.01-0.5 V/s, typically 0.1 V/s. The potentials were
standardized against the potassium ferrocyanide/ferricyanide.sup.20
and/or against Mn.sup.IIITE-2-PyP.sup.5+. All voltammograms were
reversible.
[0027] Electrospray mass spectrometry. ESMS measurements were
performed on a Micromass Quattro LC triple quadrupole mass
spectrometer equipped with a pneumatically assisted electrostatic
ion source operating at atmospheric pressure. Typically, the 0.5 mM
50% aqueous acetonitrile solutions of chloride salts of metal-free
porphyrins or their Mn(III) complexes were introduced by loop
injection into a stream of 50% aqueous acetonitrile flowing at 8
.mu.L/min. Mass spectra were acquired in continuum mode, scanning
from 100-500 m/z in 5 s, with cone voltages of 20 V and 24 V. The
mass scale was calibrated using polyethylene glycol.
[0028] Catalysis of O.sub.2.sup..cndot.- dismutation. We have
previously shown that the O.sub.2.sup..cndot.-/cytochrome c
reduction assay gives the same catalytic rate constants as does
pulse radiolysis for Mn.sup.IIITE-2-PyP.sup.5+,
{Mn.sup.IIIBVDME}.sub.2, {Mn.sup.IIIBV}.sub.2 and
MnCl.sub.2..sup.21 Therefore the convenient cytochrome c assay was
used to characterize the series of Mn(III)
N-alkylpyridylporphyrins. The xanthine/xanthine oxidase reaction
was the source of O.sub.2.sup..cndot.- and ferricytochrome c was
used as the indicating scavenger for O.sub.2.sup..cndot.-22 The
reduction of cytochrome c was followed at 550 nm. Assays were
conducted at (25.+-.1).degree. C., in 0.05 M phosphate buffer, pH
7.8, 0.1 mM EDTA, in the presence and absence of 15 .mu.g/mL of
catalase. Rate constants for the reaction of metalloporphyrins with
O.sub.2.sup..cndot.- were based upon competition with 10 .mu.M
cytochrome c, k.sub.cyt c=2.6.times.10.sup.5 M.sup.-1 s.sup.-1 as
described elsewhere..sup.21 The O.sub.2.sup..cndot.- was produced
at the rate of 1.2 .mu.M per minute. Any possible interference
through inhibition of the xanthine/xanthine oxidase reaction by the
test compounds was examined by following the rate of urate
accumulation at 295 nm in the absence of cytochrome c. No
reoxidation of cytochrome c by the metalloporphyrins was
observed.
Results
[0029] Thin Layer Chromatography. The increase in the length of the
alkyl chains is accompanied by an increase in the lipophilicity of
the compounds as indicated by the increase in the retention factor
R.sub.f (porphyrin path/solvent path) (Table 1, FIG. 2). The
apparent lag that was observed in the case of shorter chains with
Mn(III) complexes (FIG. 2B), is presumably due to their higher
overall formal charge (+5 for the Mn(III) complexes, +4 for the
ligand). As the chains lengthen, their contribution to the overall
lipophilicity increases, and eventually the n-octyl porphyrin and
its Mn(III) complex are more alike in R.sub.f than are methyl
analogues.
[0030] Uv/vis spectroscopy. Molar Absorptivities. The porphyrins
obeyed the Beer-Lambert law from 10.sup.-7 M to 10.sup.-5 M, and
the uv/vis data are given in Table 2. As the length of alkyl chains
increased from methyl to n-butyl a red shift of the Soret
absorption maxima was generally observed, as well as an increase in
the molar absorptivities, and these effects plateau beyond butyl
compound. Such trends may be understood in terms of the interplay
of porphyrin nucleus distortion (red shifts) and the
electron-withdrawing (blue shifts) effect of the N-alkylpyridyls
groups..sup.12,23
[0031] Metalation behavior and proton dissociation constants. The
rates of Mn.sup.2+ incorporation at pH .about.12.3 decreased with
an increase in chain length. The same was found for the kinetics of
Zn.sup.2+ and Cu.sup.2+ insertion into these compounds below pH 7,
where the kinetics were first order in metal and porphyrin
concentration..sup.24 Since the free-base porphyrin H.sub.2P.sup.4+
reactants were mixtures of the four atropoisomers, each isomer has
a similar metalation rate constant. As noted before for both water
soluble and insoluble porphyrins, compounds with substituents in
the ortho positions tend to metalate more slowly than derivatives
with the same groups in the meta or para positions..sup.25-34
[0032] The proton dissociation constants, K.sub.a2 and K.sub.a3 are
defined as follows:
##STR00003##
The pK.sub.a2 values for the N-alkylpyridyl series are given in
Table 1. As the alkyl chains lengthen the porphyrins become less
hydrated and the separation of charges (eq [1]) becomes less
favorable, i.e. pK.sub.a2 increases (FIG. 3, insert). FIG. 3A shows
the linear relationship between pK.sub.a2 and R.sub.f.
[0033] Equilibrium constants pK.sub.a3 for reaction [2] are 1.8 for
the meta H.sub.2TM-3-PyP.sup.4+, 1.4 for the para
H.sub.2TM-4-PyP.sup.4+, and -0.9 for ortho
H.sub.2TM-2-PyP.sup.4+..sup.4,25 While the meta and para
N-methylpyridylporphyrins are mixtures of protonated
H.sub.3P.sup.5+ and H.sub.4P.sup.6+ species in 1.0 M HCl, the ortho
substituted H.sub.2TM-2-PyP.sup.4+ to H.sub.2TnOct-2-PyP.sup.4+
compounds remain as the unprotonated free base H.sub.2P.sup.4+ in
1.0 M HCl and in 1.0 M HNO.sub.3. With ortho, meta and para
N-methylpyridylporphyrins the pK.sub.a2 increases as the pK.sub.a3
increases.
[0034] The half-lives for the acid and anion-catalyzed removal of
zinc from Zn N-methylated derivatives.sup.35 in 1.0 M HNO.sub.3
were 89 s for the meta, 165 s for the para, and 19 hours for the
ortho ZnTM-2-PyP.sup.4+. No indication of zinc loss was found
within a week for the ZnTnHex-2-PyP.sup.4+ compound..sup.36 Similar
behavior is found in 1.0 M HCl, with t.sub.1/2 ranging from 21 s
for the meta methyl to 76 hours for ZnTnOct-2-PyP.sup.4+..sup.24 In
accord are the observations that when solid MnTnHex-2-Pyp.sup.5+
was dissolved in 12 M HCl, the spectra did not change within 3
months, while over 50% of the Mn from Mn.sup.IIITM-2-PyP.sup.5+
species was lost within a month. In addition to porphyrin ring
distortion,.sup.29-32 the steric hindrance and solvation effects
imposed by the progressively longer alkyl chains may also
contribute to the differences in metalation/demetalation
behavior.
[0035] Due to their high metal-centered redox potentials, the
Mn(III) meso tetrakis ortho N-alkylpyridylporphyrins in vivo will
be readily reduced with cell reductants such as ascorbic
acid..sup.2,3,12 The reduced Mn(II) porphyrins will also be
transiently formed in the catalysis of O.sub.2.sup..cndot.-
dismutation. Therefore, we also examined the behavior of the
reduced and more biologically relevant
Mn.sup.IIIT(alkyl)-2-PyP.sup.4+ compounds. We compared the methyl,
n-hexyl and n-octyl derivatives (6 .mu.M) aerobically and
anaerobically in the presence of a 70-fold excess of ascorbic acid
(pH 7.8, 0.1 M tris buffer) and in the presence and absence of a
150-fold excess of EDTA. Under anaerobic conditions both Mn(II)
porphyrins were stable to Mn loss and porphyrin decomposition
inside 24 hours. Aerobically, .about.40% of Mn methyl but none of
the Mn n-hexyl and n-octyl compounds underwent degradation within
125 min. The absorption spectral changes indicate that the
degradation occurred through the Mn porphyrin catalyzed reduction
of oxygen by ascorbate resulting in the formation of
H.sub.2O.sub.2. The peroxide in turn causes porphyrin destruction.
These observations are consistent with previous results which
indicate that a more electron rich compound
(Mn.sup.IITM-2-PyP.sup.4+) reduces O.sub.2 faster than does a more
electron deficient species (Mn.sup.IITnOct-2-PyP.sup.4+)..sup.2,3
EDTA did not significantly influence porphyrin degradation or Mn
loss.
[0036] Electrochemistry. Cyclic voltammetry of the Mn(III)
porphyrins shows a reversible voltammogram that we ascribe to the
Mn(III)/Mn(II) redox couple. The metal-centered redox potentials,
E.sub.1/2 are in Table 1 and the representative voltammograms of
the Mn.sup.III/IITE-2-PyP.sup.5+/4+ and
Mn.sup.III/IITnHex-2-PyP.sup.5+/4+ compounds are shown in the
Supporting Material, FIG. 6. Both lipophilicity (FIG. 2B) and
E.sub.1/2 (FIG. 3B, insert) increase exponentially with the number
of CH.sub.2 groups in the alkyl chains. Consequently, the increase
in E.sub.1/2 is a linear function of R.sub.f (FIG. 3B).
[0037] Electrospray mass spectrometry. The ESMS proved to be a
valuable tool for accessing the properties of the free base
porphyrins and their Mn complexes whereby the impact of structure
on solvation, ion-pairing, redox properties,
protonation/deprotonation, dealkylation, and catalytic properties
are clearly depicted.
[0038] H.sub.2T(alkyl)-2-PyP.sup.4+. The ESMS of the metal-free
porphyrins obtained at the low cone voltage of 20 V showed dominant
molecular ions assigned to H.sub.2P.sup.4+/4 and/or its
mono-deprotonated analogue, H.sub.2P.sup.4+-H.sup.+/3 (Table 3,
FIG. 7). Negligible double deprotonation (H.sub.2P.sup.4+-2H+/2)
was noted. Only H.sub.2TM-2-PyP.sup.4+ gave rise to a
high-intensity H.sub.2P.sup.4++H.sup.+/5 peak.
[0039] The ESMS shows a pronounced decrease in solvation by
acetonitrile as the alkyl chains lengthen. Compared to the base
peak, the relative intensities of the mono-solvated molecular ions
range from 40% for methyl, 15% for ethyl, and <10% for the
higher analogues. Only with the n-hexyl and n-octyl porphyrins are
small peaks (<5%) from ions associated with chloride found.
[0040] From methyl to n-butyl, the ratio of the molecular ion to
mono-deprotonated ion peaks decreases, consistent with the trend in
pK.sub.a2. Thus, the base peak for methyl is that of the molecular
ion, while the base peak for the n-propyl and n-butyl porphyrins is
the mono-deprotonated ion. This pK.sub.a2 trend is overcome by the
higher lipophilicities of the n-hexyl and n-octyl compounds, where
roughly equal-intensity molecular ion (100%) and mono-deprotonated
ion (98%) peaks are observed. The loss of one alkyl group
(H.sub.2P.sup.4+-a.sup.+/3) was noted for all derivatives (except
for the methyl), and either no or negligible loss of a second alkyl
group (H.sub.2P.sup.4+-2a.sup.+/2) was found.
[0041] Mn.sup.IIIT(alkyl)-2-PyP.sup.5+. The ESMS of the Mn(III)
complexes was done at a lower cone voltage (20 V) than in our
previous study (30-58 V)..sup.37 Therefore, less fragmentation
occurs and more solvent-associated and ion-paired species could be
observed (Table 4, FIG. 8). Solvation and ion pairing are more
pronounced when compared with the metal-free ligands. The more
lipophilic Mn(III) porphyrins are more easily desolvated in the
electrospray ionization source. In accordance with our previous
observations, the ESMS also clearly reflects the redox properties
of these compounds..sup.37,38 The higher the E.sub.1/2 the more
reduced porphyrins are noted. Species solvated with acetonitrile or
associated with chloride were observed with both Mn(III) and Mn(II)
compounds. Two chlorides were associated only with Mn(III)
porphyrins.
[0042] In the ESMS of the n-hexyl and n-octyl porphyrins we
observed strong signals at m/z 337 and 375 that are assigned to
compounds doubly reduced either at the metal (Mn.sup.IP.sup.3+/3)
or at both the metal and porphyrin ring (Mn.sup.IIP.sup.3+-/3).
Such doubly reduced manganese porphyrins should have a higher
tendency to lose the metal, and indeed peaks for the metal-free
species were found for the n-hexyl and n-octyl derivatives, while
only traces of doubly reduced and demetalated species were found
for n-butyl.
[0043] The ESMS behavior of Mn porphyrins changes sharply once the
alkyl chains lengthen beyond butyl, as observed with corresponding
metal-free analogues. No loss of methyl groups was detected..sup.37
As the chains lengthen up to butyl the loss of an alkyl group from
Mn(III) and Mn(II) porphyrins becomes more pronounced and then the
tendency decreases with n-hexyl and n-octyl. The same trend, but of
lower intensity was noted for the loss of two alkyl groups. The
ratio of mono-chlorinated Mn(III) to mono-chlorinated Mn(II)
species decreases from methyl to n-butyl and then increases up to
n-octyl. Thus the base peak of the methyl and ethyl porphyrins
relates to Mn.sup.IIIP.sup.5++Cl.sup.-/4, while for the n-propyl
and n-butyl derivatives it relates to
Mn.sup.IIIP.sup.4++Cl.sup.-/3. Yet, with the n-hexyl, the
Mn.sup.IIIP.sup.5++Cl.sup.-/4 and Mn.sup.IIP.sup.4++Cl.sup.-/3
peaks are both of 100% intensity, and the di-chlorinated species
(Mn.sup.IIIP.sup.5++2Cl.sup.-/3) is of 86% intensity. With the
n-octyl analogue, the mono- and di-chlorinated species give rise to
100% Mn.sup.IIIP.sup.5+Cl.sup.-/4 and 89%
Mn.sup.IIIP.sup.5++2Cl.sup.-/3peaks, and the third most intense
(59%) signal relates to Mn.sup.IIP.sup.4++Cl.sup.-/3. The lack of
significant association of metal-free porphyrins with chloride
observed here and elsewhere,.sup.37 strongly supports the idea that
chloride is bound to the metal. Furthermore, at the same cone
voltage, the base peak of ortho MnTM-2-PyP.sup.5+ is the
mono-chlorinated species, which was only 35% for para isomer. This
suggests that the longer the chains, the more defined the cavity,
which can hold up to two chloride ions, and the more stable is the
Mn(III) state. While a species bearing two chlorides is hardly
noted in Mn.sup.IIITM-2-PyP.sup.5+, it is the second major peak in
the ESMS of Mn.sup.IIITnOct-2-PyP.sup.5+.
[0044] Catalysis of O.sub.2.sup..cndot.- dismutation. None of the
parent metal-free porphyrins exhibit any O.sub.2.sup..cndot.-
dismuting activity. All of the manganese compounds are potent
catalysts of O.sub.2.sup..cndot.- dismutation with log k.sub.cat
between 7.79 and 7.25. As shown in Table 1, log k.sub.cat decreases
from methyl to n-butyl and then increases, making n-octyl and
methyl of comparable antioxidant potency.
Discussion
[0045] When designing metalloporphyrin SOD mimics we are aiming at
approximating the redox properties of the enzyme active site.
Superoxide dismutases catalyse the dismutation (disproportionation)
of O.sub.2.sup..cndot.- to H.sub.2O.sub.2 and O.sub.2 at
.about.+300 V vs NHE (pH 7.0)..sup.39,40 This potential is roughly
midway (+360 mV vs NHE) between the potential for the reduction
(+890 V vs NHE).sup.41 and the oxidation of O.sub.2.sup..cndot.-
(.about.60 V vs NHE).sup.41 thus providing an equal driving force
for both half-reactions in the catalytic cycle. The
O.sub.2.sup..cndot.- dismutation by Cu,Zn-SOD occurs with catalytic
rate constant,
k.sub.cat=k.sub.red=k.sub.ox.apprxeq.2.times.10.sup.9 M.sup.-1
s.sup.-1 (log k.sub.cat.apprxeq.9.3)..sup.42-44
[0046] We previously demonstrated a structure-activity relationship
between log k.sub.cat and the metal-centered E.sub.1/2 of the
Mn(III)/Mn(II) couple for a variety of water-soluble meso
substituted porphyrins (FIG. 4A)..sup.2-4 Electron-withdrawing
substituents on the porphyrin ring shift E.sub.1/2 towards more
positive values resulting in higher values for k.sub.cat. .sup.2-4
Each 120 mV increase in E.sub.1/2 gave a 10-fold increase in
k.sub.cat,.sup.4 consistent with the Marcus equation.sup.45 for
outer-sphere electron transfer reactions (FIG. 4A). The Marcus
equation is valid as long as one of the two steps in the catalytic
dismutation cycle is
##STR00004##
rate-limiting.
[0047] On the basis of such structure-activity relationships, the
ortho isomers of Mn(III) meso tetrakis N-methyl- and
N-ethylpyridylporphyrins were tested and proved to be potent
catalysts of O.sub.2.sup..cndot.- dismutation. Their log k.sub.cat
values are 7.79 and 7.76 and they operate at potentials (+220 and
+228 V) similar to the potential of the enzyme itself. These two
metalloporphyrins also exhibit protection in in vivo models of
oxidative stress injuries..sup.14-17 We have now extended our work
to a series of Mn.sup.IIIT(alkyl)-2-PyP.sup.5+ compounds where
alkyl is methyl, ethyl, n-propyl, n-butyl, n-hexyl, and n-octyl
(FIG. 1). The significant differences in lipophilicity along the
series (FIG. 2A), with retention of catalytic potency (Table 1),
might lead to favorably selective subcellular distributions of
these new Mn.sup.IIIT(alkyl)-2-PyP.sup.5+ compounds and hence
broader their utility.
[0048] E.sub.1/2 vs pK.sub.a2. We did not expect a profound change
in E.sub.1/2 along the series based on the fact that the increase
in alkyl chain length from methyl to n-hexyl is without effect on
the basicity of alkylamines..sup.46 However, we found that the
metal-centered redox potentials varied from +220 mV for methyl to
+367 mV (vs NHE) for the n-octyl compound. Such an increase in
E.sub.1/2 may originate from progressively unshielded positive
charges at pyridyl nitrogens which would then exert stronger
electron-withdrawing effect on the coordinated Mn as the compounds
increase in lipophilicity. This reasoning is supported by the ESMS
data (Table 4, FIG. 8) which show that the susceptibility to
desolvation is accompanied by a greater preponderance of reduced
Mn(II) porphyrin ions as the alkyl chains of the Mn complexes
lengthen. We have previously reported.sup.4 that mainly electronic
effects determine the relation between the pK.sub.a of the
metal-free porphyrin and the E.sub.1/2 of the corresponding metal
complex such that the decrease in pK.sub.a3 is accompanied by a
linear increase in E.sub.1/2 (FIG. 5, insert). However as the
compounds become increasingly more lipophilic, the lack of
solvation disfavors separation of charges (higher pK.sub.a2
values), while the electron-withdrawing effects of the positively
charged pyridyl nitrogen are enhanced. Thus the electronic
pK.sub.a2 effects are overcome by solvation/steric effects
resulting in an inverted trend, i.e. the E.sub.1/2 now increases in
a linear fashion with an increase in pK.sub.a2 (FIG. 5).
[0049] Log k.sub.cat vs E.sub.1/2. Based on a previously
established structure-activity relationship for water-soluble
Mn(III) porphyrins,.sup.4 we expected the 147 mV increase in
E.sub.1/2 to be accompanied by a .about.12-fold increase in
k.sub.cat (FIG. 4A)..sup.4 We actually found that k.sub.cat
decreased from methyl to n-butyl, and then increased by the same
factor of .about.3 to n-octyl (Table 1, FIG. 4B). One explanation
is that the Mn porphyrins are solvated to different extents, as
indicated by the ESMS data, and this in turn affects the magnitude
of k.sub.cat. The trend in k.sub.cat may also be influenced by the
electrostatic/steric effects originating from the shielding of the
single positive charge on the Mn(III) center. Thus the difference
in the magnitude of lipophilicity between the metal-free ligands
(formally +4) and the Mn(III) complexes (formally +5) becomes less
noticeable as the alkyl chains get longer (Table 1). These
H.sub.2P.sup.4+ compounds of formal +4 charge behave in solution
kinetically as +1.6 to +1.8 electrolytes..sup.24 From methyl to
n-butyl, log k.sub.cat decreases almost linearly (FIG. 4B, insert).
Due to the exponential increase in E.sub.1/2 along the series of Mn
porphyrins (FIG. 3B, insert), the unfavorable electrostatic/steric
effects are in part opposed and finally overcome by the
progressively more favorable redox potentials that originate from
increased desolvation (lipophilicity). Consequently, the very
lipophilic n-octyl compound is essentially as potent an SOD mimic
as the less lipophilic methyl and ethyl derivatives.
[0050] Regan et al.sup.47 were able to uncouple the steric and
solvation effects in reactions of chloride ions with methyl- and
tert-butyl-substituted chloroacetonitrile, and showed that both
were of comparable magnitudes. Similarly, the reactivity of
N-alkylpyridylporphyrins are the result of the interplay of
electronic, steric and solvation effects, the latter dominating
with the more lipophilic members of the series.
[0051] Recent findings indicate that biologically relevant
reactions, other than O.sub.2.sup..cndot.- dismutation, can occur
at the metal center in Mn porphyrins..sup.2,3,5,7,8,48-52 The same
has been reported for the enzyme active site,.sup.20,53-57 thus
raising the complexity of the free radical chemistry and biology of
the enzymes and their mimics. Reactive oxygen and nitrogen species
are involved in direct damage of key biological targets such as
nucleic acids, proteins and fatty acids, and there is an increasing
amount of evidence that such species are also involved in the
modulation of signaling processes..sup.14,58,59 Thus, it is
important to understand the mechanisms of action of Mn porphyrins
and related compounds. Based on the electrostatic, steric,
solvation, and lipophilic effects observed in this study, we expect
the members of N-alkylpyridyl series to differ one from another in
in vivo models of oxidative stress injuries with respect to their
specificity towards reactive oxygen and nitrogen species as well as
with regard to their pharmacokinetics. Such work is in
progress.
Abbreviations
[0052] SOD, superoxide dismutase; AN, acetonitrile; DMF,
N,N-dimethylformamide; NHE, normal hydrogen electrode; TLC,
thin-layer chromatography; H.sub.2P.sup.4+, any meso tetrakis
N-alkylpyridylporphyrin ligand; Mn.sup.III/IIP.sup.4+/5+, any
Mn(III/II) meso tetrakis N-alkylpyridylporphyrin; meso refers to
the substituents at the 5,10,15, and 20 (meso carbon) position of
the porphyrin core. Mn.sup.IIIT(alkyl)-2(3,4)-PyP.sup.5+,
manganese(III) meso tetrakis(N-methyl, N-ethyl, N-n-propyl,
N-n-butyl, N-n-hexyl, N-n-octyl)pyridinium-2(3,4)-yl)porphyrin;
alkyl is M, methyl; E, ethyl; nPr, n-propyl; nBu, n-butyl; nHex,
n-hexyl; nOct, n-octyl on the pyridyl ring; 2 is the ortho, 3, the
meta and 4 the para isomer; Mn.sup.IIITM-2-PyP.sup.5+ is
AEOL-10112, and Mn.sup.IIITE-2-PyP.sup.5+ is AEOL-10113;
Mn.sup.IIIPTr(M-2-PyP.sup.4+, manganese(III) 5-phenyl-10,15,20-tris
(N-methylpyridinum-2-yl)porphyrin; Mn.sup.IIIBM-2-PyP.sup.3+,
manganese(III) meso
bis(2-pyridyl)-bis(N-methylpyridinium-2-yl)porphyrin;
Mn.sup.IIITrM-2-PyP.sup.4+,
5-(2-pyridyl)-10,15,20-tris(N-methylpyridinium-2-yl)porphyrin;
Mn.sup.IIIT(TMA)P.sup.5+, manganese(III) meso tetrakis(N,N,
N-trimethylanilinium-4-yl)porphyrin; Mn.sup.IIIT(TFTMA)P.sup.5+,
manganese(III) meso
tetrakis(2,3,5,6-tetrafluoro-N,N,N-trimethylanilinium-4-yl)poprhyrin;
Mn.sup.IIITCPP.sup.3-, manganese meso
tetrakis(4-carboxylatophenyl)porphyrin; MnTSPP.sup.3-,
manganese(III) meso tetrakis(4-sulfonatophenyl)porphyrin;
Mn.sup.IIIT(2,6-Cl.sub.2-3-SO.sub.3--P)P.sup.3-, manganese(III)
meso tetrakis(2-6-dichloro-3-sulfonatophenyl)porphyrin;
Mn.sup.IIIT(2,6-F.sub.2-3-SO.sub.3--P)P.sup.3-, manganese(III) meso
tetrakis(2,6-difluoro-3-sulfonatophenyl)porphyrin;
Mn.sup.IIIT(2,4,6-Me.sub.3-3,5-(SO.sub.3).sub.2--P)P.sup.7-,
manganese(III)
5,10,15,20-tetrakis(2,4,6,-trimethyl-3,5-disulfonatophenyl)porphyrin;
Mn.sup.IIIhematoP.sup.-, manganese(III) hematoporphyrin IX.
REFERENCES
[0053] 1. R. F. Pasternack, A. Banth, J. M. Pasternack and C. S.
Johnson, J. Inorg. Biochem. 1981, 15, 261 (b) R. F. Pasternack and
B. J. Halliwell, J. Am. Chem. Soc. 1979, 101, 1026. [0054] 2.1.
Batini -Haberle, Methods Enzymol. 2002, 349, 223. [0055] 3.1.
Spasojevi and I. Batini -Haberle, Inorg. Chim. Acta, 2001, 317,
230. [0056] 4. I Batini -Haberle, I. Spasojevi , P. Hambright, L.
Benov, A. L. Crumbliss and I. Fridovich, Inorg. Chem. 1999, 38,
4011. [0057] 5. G. Ferrer-Sueta, I. Batini -Haberle, I. Spasojevi ,
I. Fridovich and R. Radi, Chem. Res. Toxicol. 1999, 12, 42. [0058]
6. R. Kachadourian, I. Batini -Haberle and I. Fridovich, Inorg.
Chem. 1999, 38, 391. [0059] 7. J. Lee, J. A. Hunt and J. T. Groves,
J. Am. Chem. Soc. 1998, 120, 6053. [0060] 8. J. P. Crow, Arch.
Biochem. Biophys. 1999, 371, 41. [0061] 9. M. Patel and B. J. Day,
Trends Pharmacol. 1999, 20, 359. [0062] 10. (a) K. Aston, N. Rath,
A. Naik, U. Slomczynska, O. F. Schall and D. P. Riley, Inorg. Chem.
2001, 40, 1779. (b) S. Cuzzocrea, E. Mazzon, L. Dugo, A. P. Caputi,
K. Aston, D. P. Riley and D. Salvemini, Br. J. Pharmacol. 2001,
132, 19. [0063] 11. (a) S. Melov, J. Ravenscroft, S. Malik, M. S.
Gill, D. W. Walker, P. E. Clayton, D. C. Wallace, B. Malfroy, S. R.
Doctrow and G. J. Lithgow, Science, 2000, 289, 1567. (b) K. Baker,
C. Bucay Marcus, K. Huffman, H. Kruk, B. Malfroy and S. R. Doctrow,
J. Pharmacol. Exp. Ther., 1998, 284, 215. [0064] 12. I. Batini
-Haberle, L. Benov, I. Spasojevi and I. Fridovich, J. Biol. Chem.,
1998, 273, 24521. [0065] 13.1. Spasojevi , R. Menzeleev, P. S.
White and I. Fridovich, Inorg. Chem., 2002, submitted. [0066] 14.
G. B. Mackensen, M. Patel, H. Sheng, C. C. Calvi, I. Batini
-Haberle, B. J. Day, L. P. Liang, I. Fridovich, J. D. Crapo, R. D.
Pearlstein and D. S. Warner, J. Neurosci., 2001, 21, 4582. [0067]
15. J. D. Piganelli, S. C. Flores, C. Cruz, J. Koepp, I. Batini
-Haberle J. Crapo, B. J. Day, R. Kachadourian, R. Young, B. Bradley
and K. Haskins, Diabetes, 2002, 51, 347. [0068] 16. M. Aslan, T. M.
Ryan, B. Adler, T. M. Townes, D. A. Parks, J. A. Thompson, A.
Tousson, M. T. Gladwin, M. M. Tarpey, M., R. P. Patel, I. Batini
-Haberle, C. R. White and B. A. Freeman, Proc. Natl. Acad. Sci.
USA, 2001, 98, 15215. [0069] 17. (a) I. Batini -Haberle, I.
Spasojevi , I. Fridovich, M. S. Anscher and {hacek over (Z)}.
Vujaskovic, Proc of the 43.sup.rd Annual Meeting of American
Society for Therapeutics in Radiation Onciology, San Francisco
2001, 235-236. (b) Z. Vujaskovi , I. Batini -Haberle, I. Spasojevi
, T. V. Samulski, M. W. Dewhirst and M. S. Anscher, Annual Meeting
of Radiation Research Society, San Juan, Puerto Rico 2001. (c)
{hacek over (Z)}. Vujaskovi , I. Batini -Haberle, I. Spasojevi ,
Irwin Fridovich, M. S. Anscher and M. W. Dewhirst, Free Rad. Biol.
Med. 2001, S128. [0070] 18. W. R. Waud, F. O. Brady, R. D. Wiley
and K. V. Rajagopalan, Arch. Biochem. Biophys. 1975, 19, 695.
[0071] 19. T. Kaufmann, T., B. Shamsai, R. S. Lu, R. Bau and G. M.
Miskelly, Inorg. Chem. 1995, 34, 5073. [0072] 20.1. M. Kolthof and
W. J. Tomsicek, W. J., J. Phys. Chem. 1935, 39, 945. [0073] 21.1.
Batini -Haberle, I. Spasojevi , R. D. Stevens, P. Hambright, A. N.
Thorpe, J. Grodkowski, P. Neta and I. Fridovich, Inorg. Chem. 2001,
40, 726. [0074] 22. J. M. McCord and I. Fridovich, J. Biol. Chem.
1969, 244, 6049. [0075] 23.1. Batini -Haberle, S. I. Liochev, I.
Spasojevi and I. Fridovich, Arch. Biochem. Biophys. 1997, 343, 225.
[0076] 24. P. Hambright, I. Spasojevi , I. Fridovich and I. Batini
-Haberle, in preparation. [0077] 25. P. Hambright, Water-Soluble
Metalloporphyrins in The Porphyrin Handbook, K. M. Kadish, K. M.
Smith, R. Guillard, Eds. Academic Press, N.Y. 2000, Chapter 18.
[0078] 26. T. P. G. Sutter and P. Hambright, J. Coord. Chem. 1993,
30, 317. [0079] 27. L. R. Robinson and P. Hambright, Inorg. Chem.,
1992, 31, 652. [0080] 28. J. B. Reid and P. Hambright, Inorg. Chem.
1977, 16, 968. [0081] 29. M. Inamo, N. Kamiya, Y. Inada, M. Nomura
and S. Funahashi, Inorg Chem., 2001, 40, 5636. [0082] 30. P. B.
Chock and P. Hambright, J. Am. Chem. Soc. 1974, 96, 3123. [0083]
31. S. Funahashi, Y. Inada and M. Inamo, Anal. Sci. 2001, 17, 917.
[0084] 32. T. P. G. Sutter, R. Rahimi, P. Hambright, J. Bommer, M.
Kumar and P. Neta, J. Chem. Soc. Faraday Trans. 1993, 84, 495.
[0085] 33. B. Cheng, O. Q. Munro, H. M. Marques and W. R. Scheidt,
J. Am. Chem. Soc. 1997, 119, 10732. [0086] 34. R. F. Pasternack, N.
Sutin and D. H. Turner, J. Am. Chem. Soc. 1976, 98, 1908. [0087]
35. P. Hambright, T. Gore and M. Burton, Inorg. Chem. 1976, 15,
2314. [0088] 36. J, Davilla, A. Harriman, M.-G. Richoux and L. R.
Milgrom, J. Chem. Soc. Chem. Commun. 1987, 525. [0089] 37.1. Batini
-Haberle, R. D. Stevens and L Fridovich, J. Porphyrins
Phthalocyanines, 2000, 4, 217. [0090] 38. R. Kachadourian, N.
Srinivasan, C. A. Haney and R. D. Stevens, J Porphyrins
Phthalocyanines, 2001, 5, 507. [0091] 39. Vance, C. K. and Miller,
A.-F., Biochemistry, 2001, 40, 13079. [0092] 40. (a) G. D. Lawrence
and D. T. Sawyer, Biochemistry, 1979, 18, 3045. (b) W. C. Jr.
Barrette, D. T. Sawyer, J. A. Free and K. Asada, Biochemistry 1983,
22, 624. [0093] 41. Wood, P. M., Biochem. J, 1988, 253, 287. [0094]
42. Vance, C. K. and Miller, A.-F., J. Am. Chem. Soc., 1998, 120,
461. [0095] 43. R. M. Ellerby, D. E. Cabelli, J. A. Graden and J.
S. Valentine, J. Am. Chem. Soc., 1996, 118, 6556. [0096] 44. D.
Klug-Roth, I. Fridovich and J. Rabani, J. Am. Chem. Soc., 1973, 95,
2786. [0097] 45. R. A. Marcus, Annu. Rev. Phys. Chem., 1964, 15,
155. [0098] 46. CRC Handbook of Chemistry and Physics, D. R. Lide,
Editor-in-Chief, 74.sup.th Edition, 1993-1994, CRC Press, Boca
Raton. [0099] 47.1. Spasojevi , I. Batini -Haberle and I.
Fridovich, Nitric Oxide: Biology and Chemistry 2000, 4, 526. [0100]
48. (a) C. K. Regan, S. L. Craig and J. I. Brauman, Science, 2002,
295, 2245. [0101] 49. R. Shimanovich and J. T. Groves, Arch.
Biochem. Biophys., 2001, 387, 307. [0102] 50. N. Jin, J. L.
Bourassa, S. C. Tizio and J. T. Groves, Angew. Chem. Int. Ed.,
2000, 39, 3849. [0103] 51. H. Zhang, J. Joseph, M. Gurney, D.
Becker and B. Kalyanaraman, J. Biol. Chem., 2002, 277, 1013. [0104]
52. N. Motohashi and Y. Saito, Chem. Pharm. Bull., 1995, 43, 505.
[0105] 53. C. Quijano, D. Hernandez-Saavedra, L. Castro, J. M.
McCord, B. A. Freeman and R. Radi, J. Biol. Chem., 2001, 276,
11631. [0106] 54. (a) S. L. Jewet, A. M. Rocklin, M. Ghanevati, J.
M. Abel and J. A. Marach, Free Rad Biol. Med., 1999, 26, 905. (b)
S. P. A. Goss, R. J. Singh and B. Kalyanaraman, J. Biol. Chem.,
1999, 274, 28233. (c) S. I Liochev and I. Fridovich, Free Rad Biol.
Med., 199, 27, 1444. [0107] 55. (a) A. G. Estevez, J. P. Crow, J.
B. Sampson, L. Reither, J. Zhuang, G. J. Richardson, M. M. Tarpey,
L. Barbeito and J. S. Beckman, Science, 1999, 286, 2498. (b) S. I.
Liochev and I. Fridovich, J. Biol. Chem., 2001, 276, 35253. [0108]
56. S. I. Liochev and I. Fridovich J. Biol. Chem., 2000, 275,
38482. [0109] 57. E. D. Coulter, J. P. Emerson, D. M. Jr., Kurtz
and D. E. Cabelli, J. Am. Chem. Soc., 2000, 122, 11555. [0110] 58.
B. M. Matata and M. Galinanes, J. Biol. Chem., 2002, 277, 2330.
[0111] 59. (a) {hacek over (Z)}. Vujaskovi , I. Batini Haberle, M.
S. Anscher, Z. N. Rabbani, T. V. Samulski, K. Amin, M. W. Dewhirst
and Z. Haroon, Proc. of the 43.sup.rd Annual Meeting of American
Society for Therapeutics in Radiation Onciology, San Francisco
2001, 88-89. (b) {hacek over (Z)}. Vujaskovi , I. Batini -Haberle,
Z. N. Rabbani, Q.-F. Feng, S. K. Kang, I. Spasojevi , T. V.
Samulsli, I. Fridovich, M. W. Dewhirst, M. S. Anscher, Free Rad
Biol. Med. In press.
TABLE-US-00001 [0111] TABLE 1 Metal-Centered Redox Potentials
E.sub.1/2, log k.sub.cat for O.sub.2.sup..- Dismutation, and
Chromatographic R.sub.f values. E.sub.1/2.sup.c Porphyrin
R.sub.f.sup.a pK.sub.a2.sup.b mV vs NHE log k.sub.cat.sup.d
Mn.sup.IIITM-2-PyP.sup.5+ 0.09 (0.13) 10.9 +220 7.79
Mn.sup.IIITE-2-PyP.sup.5+ 0.13 (0.21) 10.9 +228 7.76
Mn.sup.IIITnPr-2-PyP.sup.5+ 0.20 (0.31) 11.4 +238 7.38
Mn.sup.IIITnBut-2-PyP.sup.5+ 0.33 (0.46) 11.7 +254 7.25
Mn.sup.IIITnHex-2-PyP.sup.5+ 0.57 (0.63) 12.2 +314 7.48
Mn.sup.IIITnOct-2-PyP.sup.5+ 0.80 (0.86) 13.2 +367 7.71
.sup.aR.sub.f (compound path/solvent path) on silica gel TLC plates
in 1:1:8 KNO.sub.3-saturated H.sub.2O:H.sub.2O:acetonitrile.
R.sub.f for the metal-free porphyrins are in parentheses.
.sup.bpK.sub.a2 determined at 25.degree. C. ionic strength 0.10
(NaNO.sub.3/NaOH). .sup.cE.sub.1/2 determined in 0.05 M phosphate
buffer (pH 7.8, 0.1 M NaCl). .sup.dk.sub.cat determined using the
cytochrome c assay, in 0.05 M phosphate buffer, pH 7.8, at (25 .+-.
1) .degree. C.
TABLE-US-00002 TABLE 2 Molar Absorptivities of Tetrads
(N-alkylpyridinium-2-yl)porphyrin chlorides and their Mn(III)
Complexes. Porphyrin .lamda..sub.nm (log .epsilon.).sup.a
H.sub.2TM-2-PyP.sup.4+ 413.2(5.32); 510.4(4.13); 544.4(3.49);
581.4(3.72); 634.6(3.13) H.sub.2TE-2-PyP.sup.4+ 414(5.33);
511(4.20); 545(3.58); 582(3.80); 635(3.38);
H.sub.2TnPr-2-PyP.sup.4+ 415(5.38); 511.5(4.24); 545(3.62);
583(3.84); 635(3.37) H.sub.2TnBut-2-PyP.sup.4+ 415(5.37);
511(4.24); 544(3.60); 583(3.84); 636(3.39)
H.sub.2TnHex-2-PyP.sup.4+ 415.5(5.34); 510.5(4.24); 543(3.62);
584.5(3.84); 638(3.43) H.sub.2TnOct-2-PyP.sup.4+ 416.5(5.31);
510(4.25); 542(3.59); 585(3.82); 639.5(3.43)
Mn.sup.IIITM-2-PyP.sup.5+ 363.5(4.64); 411(4.27); 453.4(5.11);
499(3.66); 556(4.03); 782(3.15) Mn.sup.IIITE-2-PyP.sup.5+
363.5(4.68); 409(4.32); 454(5.14); 499(3.75); 558(4.08); 782(3.26)
Mn.sup.IIITnPr-2-Pyp.sup.5+ 363(4.70); 411(4.37); 454(5.21);
498(3.81); 559(4.12); 782(3.35) Mn.sup.IIITnBut-2-PyP.sup.5+
364(4.70); 410(4.35); 454(5.23); 498(3.83); 559(4.14); 781(3.33)
Mn.sup.IIITnHex-2-PyP.sup.5+ 364.5(4.70); 415(4.57); 454.5(5.21);
507(3.85); 560(4.12); 780(3.30) Mn.sup.IIITnOct-2-PyP.sup.5+
364(4.72); 414(4.44); 454.5(5.24); 500.5(3.84); 559.5(4.14);
781(3.25) .sup.aThe molar absorptivities were determined in water
at room temperature.
TABLE-US-00003 TABLE 3 EIectrospray Mass Spectrometry Results for
H.sub.2T(alkyl)-2-PyP.sup.4+ Compounds..sup.a m/z Species.sup.b M E
nPr nBu nHex nOct H.sub.2P.sup.4+/4 169 184 198 212 239 268
H.sub.2P.sup.4++AN/4 180 194 208 222 250 H.sub.2P.sup.4++2AN/4 190
H.sub.2P.sup.4+-H.sup.+/3 226 245 263 282 319 357
H.sub.2P.sup.4+-H.sup.++AN/3 240 258 278
H.sub.2P.sup.4+-H.sup.++H.sub.2O/3 288
H.sub.2P.sup.4+-H.sup.++Cl.sup.-/2 496 H.sub.2P.sup.4+-a.sup.+/3
235 249 263 291 319 H.sub.2P.sup.4+-a.sup.+-H.sup.+/2 352 374 394
436 H.sub.2P.sup.4+-a.sup.++H.sub.2O/3 255
H.sub.2P.sup.4+-2a.sup.+/2 352 366 H.sub.2P.sup.4++H.sup.+/5 136
H.sub.2P.sup.4++H.sup.++AN/5 143 H.sub.2P.sup.4++H.sup.++2AN/5 152
H.sub.2P.sup.4++H.sup.++2Cl.sup.-/3 343 381
H.sub.2P.sup.4++2H.sup.++2Cl.sup.-/4 286 H.sub.2P.sup.4+-2H.sup.+/2
339 367 395 423 479 .sup.a~0.5 mM solutions of H.sub.2P.sup.4+ in
1:1 acetonitrile:water, 20 V cone voltage. .sup.bAN denotes
acetonitrile and a is an alkyl group.
TABLE-US-00004 TABLE 4 Electrospray Mass Spectrometry for
Mn.sup.IIIT(alkyl)-2-PyP.sup.5+ Porphyrins..sup.a m/z Species.sup.b
M E nPr nBu nHex nOct Mn.sup.IIIP.sup.5++5 146 157
Mn.sup.IIIP.sup.5++AN/4 155 166 177 188 Mn.sup.IIIP.sup.5++2AN/5
163 174 185 196 Mn.sup.IIIP.sup.5++3AN/5 171 182 193 205
Mn.sup.IIIP.sup.5++4AN/5 179 190 213 Mn.sup.IIIP.sup.5++5AN/5 187
198 Mn.sup.IIIP.sup.5++6AN/5 195 Mn.sup.IIIP.sup.5++H.sub.2O/5 150
Mn.sup.IIIP.sup.5++Cl.sup.-/4 192 206 234 262 290
Mn.sup.IIIP.sup.5++2Cl.sup.-/3 267 286 305 323 361 398
Mn.sup.IIIP.sup.5++2Cl.sup.-+AN/4 202 216 230 244 272
Mn.sup.IIIP.sup.5+-a/4 200 Mn.sup.IIIP.sup.5+-a+AN/4 200 221 242
Mn.sup.IIIP.sup.5+-a+2Cl.sup.-/3 264 279 293 321 349
Mn.sup.IIIP.sup.5+-2a/3 243 252 262 299 Mn.sup.IIIP.sup.5+-2a+AN/3
275 294 Mn.sup.IIP.sup.4+/4 183 197 211 281 Mn.sup.IIP.sup.4++AN/4
193 207 221 235 263 Mn.sup.IIP.sup.4++2AN/4 204
Mn.sup.IIP.sup.4+/Cl.sup.-/3 255 274 293 312 349 387
Mn.sup.IIP.sup.4+-a/3 253 266 281 309 337
Mn.sup.IIIP.sup.5+-Mn.sup.3++H.sup.+/3 281 319 357
Mn.sup.IIP.sup.-3+/3 or Mn.sup.IP.sup.3+/3 294 337 375 .sup.a~0.5
mM solutions of Mn.sup.IIIP.sup.5+ in 1:1 acetonitrile:water, 20 V
cone voltage. .sup.bAN denotes acetonitrile and a is an alkyl
group.
* * * * *