CA2359116C - Substituted porphyrins - Google Patents

Abstract

The present invention relates, in general, to a method of modulating physiological and pathological processes and, in particular, to a method of modulating cellular levels of oxidants and thereby processes in which such oxidants are a participant. The invention also relates to compounds and compositions suitable for use in such methods.

Description

SUBSTITVTED PORPHYRlr"S

TECWC AL FIELD

The present invention relates, in general, to a method of modulatina physiological and pathological processes and, in particular, to a method of modulatinc, cellular levels of oxidants and therebv processes in which such oxidants are a participant. The invention also relates to compounds and compositions suitable for use in such methods.

BACKGROL~D
Oxidants are produced as part of the normal metabolism of all cells but also are an important component of the pathoeenesis of manv disease processes.
Reactive oxvgen species, for example, are critical eleme^.ts of the pathoaenesis of diseases of the luna, the cardiovascular svstem, the sastrointestinal svstem, the central nervous svstem and skeletal muscle. Oxvzen free radicals also plav a role in modulatinc, the effects of nitric oxide (N0-). In this conte:ct. thev contribute to the pathogenesis of vascular disorders, inflammatorv diseases and the aging process.
A critical balance of defensive enzvmes aQainst oxidants is required to maintain normal cell and organ function. Superoxide dismutases (SODs) are a familv of inetalloenzvmes that catalvze the-intra- and extracellular conversion of 02- into H102 plus 02, and represent the first line of defense auainst the detrimental effects of superoxide radicals. Nlammals produce three distinct SODs.
One is a dimeric copper- and zinc-containing enzvme (CuZn SOD) found in the cytosol of all cells. A second is a tetrameric manQanese-containing SOD (N1n SOD) found within mitochondria, and the third is a tetrameric, alycosylated, copper-and zinc-containing enzyme (EC-SOD) found in the extrwellular fluids and bound to the extracellular matrix. Several other important antioxidant enzymes are known to exist within cells, including catalase and glutathione peroxidase. While e:ctracellular fluids and the e:ctracellular matris contain only small amounts of these enzymes, other extracellular antioxidants are also known to be present, including radical scavengers and inhibitors of lipid peroxidation, such as ascorbic acid, uric acid, and a-tocopherol (Halliwell et al, Arch. Biochem. Biophys. 280:1 (1990)).

The present invention relates generally to low molecular weight porphyrin compounds suitable for use in modulatin; intra- and ex=cellular processes in which superoxide radicals, or other oxidants such as hvdroQen peroxide or peroxvnitrite, are a participant. The compounds and methods of the invention find application in various physiologic and pathologic processes in which oxidative stress plays a role.

St'MN1ARY OF THE ItitiEINMON

The present invention relates to a method of modulating intra- or extracellular levels of oxidants such as superoxide radicals, hydrogen peroxide, peroxynitrite, lipid peroxides, hydro:cyl radicals and thiyl radicals. ivlore particularly, the invention relates to a method of modulating normal or pathological processes involvina superoxide radicals. hvdrozen peroxide, nitric oxide or pero:cynitrite using low molecular weight antioxidants, and to methine (ie, meso) substituted porphyrins suitable for use in such a method.

2 More specifically, the present invention relates to a compound of formula Rt ~ I \
NH N
Ra R2 N NH

or pharmaceutically acceptable salt thereof, wherein R i and R3 are the same and are:

Y X I
N N X S N
CF3, COZ X, +D , --~~ + I , --~\' N+ _ X --< J , N N x N
I x X x ~ X RS X RS X N
~+ , ~ (N , N , ~ or N }
N S X N
x R5 R5 R2 and R4 are the same and are:

x X
S N S
N N N X
-(, ~ + ---\ ~X N+_ X
+N S N N N
x x X x 2a X X Nl I' X N' N' ~ I , NJ N ' N+ -- N, N
I X
X R$ Rs Y is halogen or -COzX, and X is the same or different and is an alkyl group and each R5 is the same or different and is H or alkyl, N
wherein when R, and R3 are -H, R2 and R4 are not ---~~ ], or X
N
when R, and R3 are -H and R2 and R4 are --~ said compound is N
i X
complexed with a metal selected from the group consisting of manganese, iron, copper, cobalt and nickel.
The present invention further relates to the use of such compound for (a) protecting cells from oxidant-induced toxicity;
(b) treating a patient suffering from a condition that result from or that is exacerbated by oxidant-induced toxicity;
(c) treating a pathological condition of a patient resulting from degradation of NO or a biologically active form thereof;
(d) treating a patient for an inflammatory disease; or (e) treating a patient for an ischemic reperfusion injury.
The present invention also relates to a method of protecting cells from oxidant-induced toxicity comprising contacting said cells ex vivo with a protective amount of a compound of formula 2b Ri NH N
Ra / \ R2 N NH

or pharmaceutically acceptable salt thereof, wherein Ri and R3 are the same and are:

Y X X
I N I N x S~ N
H, -CF3, -C42-X, +I
D ~X
N N N+-X N
I
x x x RS RS X
X x x N

N+ N N or -~~ N
s J X N
N

R2 and R4 are the same and are:

x X
S N ~ N X N~ S

S N N N N+- X
X
x X X x X X x N I
' N+, N, N
N
- I~ N N+ r --\
N ~ , x N
x R5 R5 2c Y is halogen or -COZX, and X is the same or different and is an alkyl group and each R5 is the same or different and is H or alkyl, so that said protection is effected.

The present invention also relates to the use of a compound of the formula R;

NH N
Ra R2 N NH

or pharmaceutically acceptable salt thereof, wherein R i and R3 are the same and are:

Y X X
N N x s N
-CF3, -C42-X, - , 1, --(, +D , --C, + , , -- ~~ N+T
N N X N
I }( X
x X X x N
* N N' N
f1l~~ ~ N or -~~ N N SJ X N

I x R5 Rs R2 and R4 are the same and are:

2d x X
S N N x N S
-N X+~ \+~
~ N+-X
J
+ S N N x N
I X
X X x Rs Rs X
+X
X X N
N' N N+ 01 J
N~ N N , X NN
I
X Rs Rs Y is halogen or -CO2X, each X is the same or different and is an alkyl group and each R5 is the same or different and is H or alkyl, in the preparation of a medicament for (a) protecting cells from oxidant-induced toxicity;
(b) treating a patient suffering from a condition that result from or that is exacerbated by oxidant-induced toxicity;

(c) treating a pathological condition of a patient resulting from degradation of NO or a biologically active form thereof;
(d) treating a patient for an inflammatory disease; or (e) treating a patient for an ischemic reperfusion injury.
Objects and advantages of the present invention will be clear from the description that follows.

2e BRIEF DESCRIPTIOy OF THE DRAWING

Figures lA-C show the structures of certain compounds of the invention.
The SOD activity values were determined using the method of McCord and Fridovich, J. Biol. Chem. 244:6049 (1969). The catalase values were determined using the method of Day et al, Arch. Biochem. Biophys. 347:256 (1997). The TBARS values were obtained as follows:

Homogenates Frozen adult Sprague-Dawley rat brains. livers and mouse lunLys (Pel-Freez, Rogers, AR) were homogenized with a polytron (Turrax T25, Germany) in 5 volumes of ice cold 50 mM potassium phosphate at pH 7.4. Homogenate protein concentration was determined with the Coomassie Plus protein assay (Pierce, Rockford, IL) using bovine serum albumin as a standard. The homogenate volume was adjusted with buffer to aive a final protein concentration of 10mJmi and frozen as aliquots at -80 C.

Oxidation of homogenates Microfuge tubes (1.5 ml) containing 0.2 ml of homogenate (0.2 mg protein) and various concentrations of antioxidant were incubated at 37 C for 15 minutes.
Oxidation of the rat brain homoaenate was initiated by the addition of 0.1 ml of a freshly prepared stock anaerobic solution containing ferrous chloride (0.25 m1v1) and ascorbate (1-mM). Samples were placed in a shaking water bath at 37 C for 30 minutes (final volume 1 ml). The reactions were stopped by the addition of 0.1 L of a stock butylated hydroxytoluene (60 m:I) solution in ethanol.
Lipid peroxidation measurement The concentration of thiobarbituric acid reactive species (TBARS) in rat brain homogenates was used as a index of.lipid peroxidation. Malondialdehyde

3 standards were obtained by adding 8.2 L of 1,I3,3-tetramethoxypropane in 10 mI
of 0.01 N HCI and mixing for 10 minutes at room temperature. This stock was further diluted in water to give standards that ranged from 0.25 to 25 M.
Samples or standards (200 L) were acidified with 200 L of 0.2 M stock of phosphoric acid in 1.5 ml locking microft:ge tubes. The color reaction was initiated by the addition of 25 L of a stock thiobarbituric acid solution (0.11N1) that was miYed and then placed in a 90 C heating block for 30 minutes. TBARS were extracted with 0.5 ml of n-butanol bv vortexing for 3 minutes and chillinc, on ice for 1 minute. The samples were then centrifuged at 12,000 x g for 3 minutes and a 150 L aliquot of the n-butanol phase was placed in each well of a 96-well plate and read at 53 _5 nm in a Thermomax platereader (-Molecular Devices, Sunnydale. CA) at 25'C. Sample absorbances were converted to IvIDA equivalences ( M) by extrapolation from the MDA standard curve. None of the antioxidants at concentrations employed in these studies affected the reaction of vIDA standards with thiobarbituric acid.

Statistical analyses Data were presented as their means = SE. The inhibitory concentration of antioxidants that decreased the degree of lipid peroxidation by 50% (ICSO) and respective 95% confidence intervals (CI) were determined bv fittina a sismoidal curve with variable slope to the data (Prian, GraphPad. San Diego, CA). (See also Braughler et_al, J. Biol. Chem. 262:10438 (1987); Kikugawa et al, Anal.
Biochetn.
202:249 (1992).) Fiaure 2 shows the data obtained from a studv involvinQ treatment of bronchopulmonary dvsplasia usina Aeol-V.

4

5 PCT/US00/02062 DETAILED DESCRIPTION OF THE IIWENTION

The present invention relates to methods of protecting against the deleterious effects of oxidants, particularly. superoxide radicals, hydrogen peroxide and peroxvnitrite, and to methods of preventing and treating diseases and disorders that involve or result from oxidant stress. The invention also relates methods of modulating biological processes involvinc, oxidants, including superoxide radicals, hydrogen peroxide, nitric oxide and peroxynitrite. The invention further relates to compounds and compositions, including low molecular weiQht antioxidants (eg mimetics of scavensers of reactive oxygen species. including mimetics of SODs, catalases and peroxidases) and formulations thereof, suitable for use in such methods.

Mimetics of scavensers of reac:ive oxygen species appropriate for use in the present methods include methine (ie meso) substituted porphines, or pharmaceutically acceptable salts thereof (ea chloride or bromide salts). The invention includes both metal-free and metal-bound porphines. In the case of metal-bound porphines, manganic derivatives of methine (meso) substituted porphines are preferred, however. metals other than manganese such as iron (II
or III), copper (I or II), cobalt (II or III), or nickel (I or II), can also be used. It will be appreciated that the metal selected can have various valence states, for e:cample, man?anese II; III or V can be used. Zinc (II) can also be used even though it does not underso a valence chanze and therefore will not directlv scavenge superoxide.
The choice of the metal can affect selectivity of the oxy~en species that is scavenged. Iron-bound porphines. for example, can be used to scavenge vO-while manQanese-bound porphines scavenge N0= less well.

The mimetics of the present invention are of the Formula I:

/ ~ ~ \
NH N
R4 / \ ~
~N ~ \
\ ~ -,..

or pharmaceuticaliv acceptable salt thereof wherein:
R, and R, are the same and are:

Y X X
N N x g N
_1-,-CF3,-CO2_x, - , , ,~ , , ---{, ~ , ----~ ---~ ~ ~
N+"x N
N N X ~
x X x x RS X RS x N
X
N+ lN N N or N, i sJ .- N x N.N
X s R5 R: and R~ are the same and are :

x X
S N S
N N N X
" ~ ' ~~ ' ---~~ + ~ - --~~+ N+-X
+N S N' N X N

x x X x

6 RS Rs X
X N
<+x ~NN 1 ~ ~
-{, N N -~ ,X N,N

Y is halogen or -COZX, each X is the same or different and is an alkyl and each R, is the same or different (preferably the same) and is H or alkyl.

Preferably, R, and R, are the same and are:

X ~{
Y IX N X X N
-H, -CF3, -COz'X, ---,+ D I - -or ---C~ I I
~ N N X N N
x X x R, and R, are the same and are :

N~ or N N
I X x X X
Rs Y is -F or -CO2X

7 WO 00/43395 PCr/US00/02062 each X is the same or different and is an alkyl (preferably, C, alkyl, e.;., methyl or ethyl) and each R, is the same or different (preferably the same) and is H
or alkyl (preferabiy, C, alkyl, e.;. -CH3 or -CH.CH3).
X
Most preferabiv. Rõ R,, R, and R, are the same and are ~( or X N

( and each X is the same or different and is C, aikvl, advantn~eouslv.
N X .
x methyl or ethyl, particulariv, methvl.

Specific examples of mimetics of rhe invention are shown in Fi2ure 1.
together with activity data.

In addition to the methine (meso) substituents described above. one or more of the pyrrole rings of the porphyrin of Formula I can be substituted at anv or all beta carbons, ie: 2, 3, 7, 8, 121, 1;. 17 or 18. Such substituents, desisnated P. can be hydroQen or an electron withdrawzng group. for example, each P can, independentlv, be a NO, group. a halo8en (ea Cl, Br or F), a nitrile group. a vinyl group. or a formvI group. Such substituents alter the redox potential of the porphyrin and thus enhance its abilitv to scavenge oxyLyen radicals. For example.
there can be 1, 2, 3, 4, 5, 6, 7, or 8 halogen (eg Br) substituents (preferably. 1-4). the remainina P's advantageousiv bein2 hvdroQen. When P is formvl, it is preferred that there not be more than ?(on non-adjacent c.arbons), more preferablv, 1, the remaining P's preferably being hvdroeen. When P is NOZ, it is preferred that there not be more than 4 (on non-adjacent carbons), more preferably, I or 2, the remaining P's being hydrogen. _ W'here isomers are possible, all such isomers of the herein described mimetics are within the scope of the invention.

8 Mimetics preferred for use in the present methods can be selected by assaying for SOD, catalase and/or peroxidase activity. Mimetics can also be screened for their ability to inhibit lipid peroxidation or scavenge ON00- (as determined by the method of Szabo et al. FEBS Lett. 381:82 (1996)).
SOD activity can be monitored in the presence and absence of EDTA using the method of McCord and Fridovich (J. Biol. Chem. 244:6049 (1969)). The efficacy of a mimetic can also be determined by measuring the effect of the mimetic on the aerobic growth of a SOD null E. coli strain versus a parent strain.
Specifically, parental E. coli (A.B 11 _57) and SOD null E. coli. (JI132) can be 2row-n in ti19 medium containinQ 0.2% casamino acids and 0.2% glucose at pH 7.0 and 37 C; grow-th can be monitored in terms of turbiditv followed at 700 nrrm.
Tnis assav can be made more selective for SOD mimetics bv omittins the branched chain, aromatic and sulphur-containing amino acids from the medium (glucose minimal medium (N19), plus 5 essential amino acids).

Eff'icacy of active mimetics can also be assessed by determining their ability to protect mammalian cells against methvlvioloQen (paraquat)-induced toxicity.
SpecificaIlv, rat L2 cells srown as described below and seeded into 24 well dishes can be pre-incubated with various concl-ntrations of the SOD mimetic and then incubated with a concentration of methylviologen previously shown to produce an LC75 in control L2 cells. Efficacy of the mimetic can be correlated with a decrease in the methylviologen-induced LDH release (St. Clair et al, FEBS Lett. 293:199 (1991)).

The efficacy of SOD mimetics can be tested in vivo with mouse and/or rat models using both aerosol administration and parenteral injection. For example, male Balbic mice can be randomized into 4 groups of 8 mice each to form a standard 2X2 continaencv statistical model. Animals can be treated with either paraquat (40 mg/kg, ip) or saline and treated ~N1th SOD mimetic or vehicle control.

9 Lung injury can be assessed 48 hours after paraquat treatment by analysis of bronchoalveolar lavage fluid (BALF) damage parameters (LDH, protein and %
PNIN) as previously described (Hampson et al, Tox. Appi. Pharm. 98:206 (1989);
Day et al, J. Pharm. 'lvlethods 24:1 (1990)). Lungs from 2 mice of each group can be instillation-fixed with 4% paraformaldehyde and processed for histopathology at the light microscopic level.

Catalase activity can be monitored by measuring absorbance at 2=I0nm in the presence of hydrogen peroxide (see Beers and Sizer, J. Biol. Chem. 195:133 (1952)) or bv measuring oxvaen evolution with a Clark oxygen electrode (Del Rio et al, Anal. Biochem. 80:409 (1977)).

Peroxidase activity can be measured specnophotometricallv as previously described by Putter and Becker: Pe:oxida<es. In: Methods of Enzymatic Analysis, H.U. Bergmever (ed.), Verlag Chemie, w'einheim, pp. 286-292 (1983). Aconitase activiry can be measured as described by Gardner and Fridovich (J. Biol. Chem.
266:19328 (1991)). The selective, reversible and SOD-sensitive inactivation of aconitase by known O_- generators can be used as a marker of intracellular O,' generation. Thus, suitable mimetics can be selected bv assaying for the abilitv to protect aconitase activiry.

The ability of mimetics to inhibit lipid peroxidation can be assessed as described by Ohkawa et al (Anal. Biochem. 95:351 (1979)) and Yue et al (J.
Pharmacol. Exp. Ther. 263:92 (1992)). Iron and ascorbate can be used to initiate lipid peroxidation in tissue homogenates and the formation of thiobarbituric acid reactive species (TBARS) measured.

Active mimetics can be tested for toxicitv in mammalian cell culture by measuring lactate dehydrogenase (LDH) release. Specifically, rat L2 cells (a lung Type II like cell (Kaighn and Douglas, J. Cell Biol. 59:160a (1973)) can be srown in Ham's F-12 medium with 1091'0 fetal calf serum supplement at pH 7.4 and 37 C;

cells can be seeded at equal densities in 24 well culture dishes and grown to approximatelv 90% confluence; SOD mimetics can be added to the cells at loa doses (eg micromolar doses in minimal :ssential medium (IMEM)) and incubated for 24 hours. Toxicity can be assessed by morphology and by measuring the release of the cytosolic injury marker, LDH (eg on a thermokinetic plate reader), as described by Vassault (In: Methods of Enzymatic Analysis, Ber;meyer (ed) pp. 113-26 (1983); oxidation of NADH is measured at 340 nm).

The mimetics of the present invention are suitable for use in a varie^r of methods. The compounds of Formula I, particularly the metal bound forms (advantageouslv. the man-anese bound forms), are characterized bv the abilir.1 to inhibit lipid peroxidation. Accordinaly. these compounds are preferred for use in the treatment of diseases or disorders associated with elevated levels of lipid peroxidation. The compounds are further preferred for use in the treatment of diseasCs or disorders mediated by oxidative stress. Inflammatory diseases are examples, includinc, asthma. inflammatory bowel disease, arthritis and vasculitis.

The compounds of the invention (advantageously, metal bound forms thereof) can also be used in methods desi2ned to rezulate ti0- levels by targeting the above-described porphines to strategic locations. iv0- is an intercellular sijnal and, as such, NO- must traverse the extracellular matrix to exert its effects.
NO-, however, is highly sensitive to inactivation mediated bv 0,' present in the extracellular spaces. The methine (meso) substituted porphyrins of the invention can increase bioavalabilitv ofti0- bv preventing its de2radation bv 0,' .

The present invention relates, in a further specific embodiment, to a method of inhibiting production of superoxide radicals. In this embodiment, the mimetics of the invention (particularlv.metal bound forms thereof) are used to inhibit oxidases, such as xanthine oxidase, that are responsible for production of superoxide radicals. The ability of a mimetic to protect mammalian cells from xanthine!xanthine oxidase-induced injury can be assessed, for example, by growing rat L2 cells in 24-well dishes. Cells can be pre-incubated with various concentrations of a mimetic and then xanthine oxidase (XO) can be added to the culture along with Yanthine (X). T'ne appropriate amount of XO/X used in the study can be pre-determined for each cell line bv performing a dose-response curve for injury. X/XO can be used in an amount that produces approximately an LC7; in the culture. Efficacy of the mimetic can be correlated with a decrease in XO/~C-induced LDH release.

The mimetics of the invention (particularly, metal bound forms thereof ) can also be used as catalvtic scavenoers of reactive oxvoen species to protect against ischemia reperfusion injuries associated with mvocardial infarction, coronan=
bypass suraerv, stroke, acute head trauma, orsan reperfusion followin-transplantation, bowel ischemia. hemorrhaQic shock, pulmonary infarction, surgical occlusion of blood flow, and soft tissue injury. The mimetics (particularlv.
metal bound forms) can further be used to protect aaainst skeletal muscle reperfusion injuries. The mimetics (particularly, metal bound fornns) can also be used to protect azainst damage to the eve due to sunlight (and to the skin) as well as -laucoma.
cataract and macular deueneration of the eye. The mimetics (particularlv, metal bound forms) can also be used to treat burns and skin diseases, such as dermatitis, psoriasis and .other inflammatory skin diseases. Diseases of the bone are also amenable to treatment with the mimetics. Further, connective tissue disorders associated with defects in collasen synthesis or demdation can be expected to be susceptible to treatment with the present mimetics (particularly, metal bound forms), as should the generalized deficits of aQing. Liver cirrhosis and renal diseases (including glomerula nephritis, acute tabular necrosis, nephroderosis and dialysis induced complications) are also amenable to treatment with the present mimetics (particularly, metal bond forms thereof).

t'_ The mimetics of the invention (particularlv, metal bound forms) can also be used as catalvtic scavengers of reactive oxygen species to increase the very limited storage viability of transplanted hearts, livers, lungs, kidneys, skin and other organs and tissues. The invention also provides methods of inhibiting damage due to autoxidation of substanc.-s resultina in the formation of OZ' including food products, pharmaceuticals, stored blood, etc. To effect this end, the mimetics of the invention are added to food products, pharmaceuticals. stored blood and the like, in an amount sufficient to inhibit or prevent oxidation damage and thereby to inhibit or prevent the degradation associated with the autoxidation reactions. (For other uses of the mimetics of the invention, see USP 5.227.405). The amount of mimetic to be used in a particular treatment or to be associated with a particular substance can be determined by one skilled in the art.

The mimetics (particularly, metal bound forms) of the invention can further be used to scavenue hvdrocen peroxide and thus protect aQainst formation of the highly reactive hydroxyl radical by interfering with Fenton chemistry (Aruoma and Halliwell, Biochem. J. 241:273 (1987): NMello Filho et al. Biochem. J. 218:273 (1984); Rush and Bielski. J. Phys. Chem. 89:5062 (1985)). The mimetics (particularly. metal bound forms) of the invention can also be used to scavenge peroYvnitrite, as demonstrated indirectlv bv inhibition of the oxidation of dihydrorhodamine 123 to rhodamine 123 and directly by accelerating peroxvnitrite degradation by stop flow analysis.

Further examples of specific diseases disorders appropriate for treatment using the mimetics of the present invention, advantaseouslv, metal bound forms, include diseases of the cardiovascular system (including cardiomyopathy, ischemia and atherosclerotic coronary vascular disease), central nervous svstem (including AIDS dementia, stroke, amyotrophic lateral sclerosis (AI.S), Parkinson's disease and HuntinQton's disease) and diseases of the musculature (including diaphramic diseases (eg respiratory fatigue in chronic obstructive pulmonary disease, cardiac fatigue of congestive heart failure, muscle weakness syndromes associated with myopathies, ALS and multiple sclerosis). Many neurologic disorders (including epilepsy, stroke, Huntington's disease, Parkinson's disease, ALS, Alzheimer's and AIDS dementia) are associated with an over stimulation of the major subtype of glutamate receptor, the NLIMDA (or N-methyl-D-aspartate) subtype. On stimulation of the NMDA receptor, excessive neuronal calcium concentrations contribute to a series of membrane and cvtoplasmic events Ieading to production of oxygen free radicals and nitric oxide (N'O-). Interactions between oxyaen free radicals and N0-have been shown to contribute to neuronal cell death. Well-established neuronal cortical culture models of N-N1DA-toxicitv have been developed and used as the basis for drug development. In these same systems. the mimetics of the present invention inhibit ti'MDA induced injury. The formation of O." radicals is an obligate step in the intracellular events culminatins in excitototiic death of cortical neurons and fi.u-ther demonstrate that the mimetics of the invention can be used to scavenge O," radicals and therebv serve as protectants aaainst excitotoxic injury.

The present invention also relates to methods of treatina AIDS. The Nf Kappa B promoter is used by the HIV virus for replication. This promoter is redox sensitive, therefore, an oxidant can regulate this process. This has been shown previously for two metalloporphyrins distinct from those of the present invention (Song et al, Antiviral Chem. and Chemother. 8:85 (1997)). T'ne invention also relates to methods of treating systemic hypertension, atherosclerosis, edema, septic shock, pulmonary hypertension, including primary pulmonarv hvpertension.
impotence, infertility, endometriosis, premature uterine contractions, microbial infections, gout and in the treatment of Type I or Type II diabetes mellitus.
The mimetics of the invention (particularly, metal boundfottns) can be used to ameliorate the toxic effects associated with endotoxin, for example, by preserving vascular tone and preventing multi-organ system damage.
As indicated above, inflammations, particularly inflammations of the lung, are amenable to treatment using the present mimetics (particularly, metal bound forms) (particularly the inflammatory based disorders of emphysema, asthma, A.R.DS including oxygen toxicity, pneumonia (especially kTDS-related pneumonia), cystic fibrosis, chronic sinusitis, arthritis and autoimmune diseases (such as lupus or rheumatoid arthritis)). Pulmonary fibrosis and inflammatory reactions of muscles, tendons and li9aments can be treated usinz the present mimetics (particularly metal bound forms thereof). EC-SOD is localized in the interstitial spaces surrounding airways and vasculature smooth muscle cells. EC-SOD and 0." mediate the antiinflammatory - proinflammatorv balance in the alveolar septum. NO-released by alveolar septal cells acts to suppress inflammation unless it reacts with 0_' to form ONOO-. By scavenQin~ 0,', EC-SOD tips the balance in the a:veolar septum aeainst inflammation_ Significant amounts of ON00- will form only when EC-SOD is deficient or when there is 2reatly increased 0_' release.
i'vfimetics described herein can be used to protect against destruction caused bv hyperoxia.
The invention further relates to methods of treatinQ memory disorders. It is believed that nitric oxide is a neurotransmitter involved in lona-term memor-y potentiation_ Using an EC-SOD knocked-out mouse model (Carlsson et al. P:oc.
Natl. Acad. Sci. USA 92:6264 (1995)), it can be shown that learning impairment correlates with reduced superoxide scavenging in extracellular spaces of the brain.
Reduced scavengin- results in hiaher extracellular 0; levels. 0_' is believed :o react with nitric oxide thereby preventing or inhibiting nitric oxide-mediated neurotransmission and thus loncy-tetTn memory potentiation. The mimetics of the invention, particularly, metal bound forms, can be used to treat dementias and memory/learnincy disorders.

The availability of the mimetics of the invention also makes possible studies of processes mediated by 0_', hydrogen peroxide, nitric oxide and perox=mitrite.
The mimetics described above, metal bound and metal free forms, can be formulated into pharmaceutical compositions suitable for use in the present methods. Such compositions include the active agent (mimetic) together with a pharmaceutically acceptable carrier, excipient or diluent. T'ne composition can be present in dosage unit form for example, tablets, capsules or suppositories.
The composition can also be in the form of a sterile solution suitable for injec:ion or nebulization. Compositions can also be in a form suitable for opthalmic use.
The invention also includes compositions formulated for topical administration.
such compositions takinsz the form, for example. of a Iotion. cream. gel or ointment. The concentration of active agent to be included in the composition can be selected based on the nature of the aQent. the dosage regimen and the result souaht.
The dosaize of the composition of the invention to be administered can be determined without undue experimentation and will be dependent upon various factors including the nature of the active agent (including whether metal bound or metal free), the route of administration, the patient, and the result sought to be achieved. A suitable dosase of mimetic to be administered IV or topically can be expected to be in the range of about 0.0 1 to 50 mg/k~/dav, preferablv, 0.1 to mJkgJday. For aerosol administration, it is expected that doses will be in the range of 0.001 to 5.0 mQ,kJdav, preferably, 0.01 to 1 mQ/lcg/dav. Suitable doses of mimetics will varv, for example, with the mimetic and w~ith the result sought.

Certain aspects of the present invention will be described in greater detail in the non-limiting Examples that follow. (The numbering of the compounds in Example I is for purposes of that Example only.) EXAMPLE I

Syntheses I. [5,15-Bis(4-carbomethoayphenyl)-10,20-(thiazol-5-yi)porphyrinatoj-manganese(III) Chloride (5).

N=1 S

,0 + / \ TFa \ \ ~ ~
OHC S H CH2CI, NH HN

CO'NiC
o-CHO

TFA. CH,C1,_ DDQ
CO2Me CO"Mz " s ~ n N N ~InCI2, DNtF ~ s ~ NH N N
N M
- N SJ ~ A - 'N' H~I ` S
CO,Me CO,I'v te 1. meso-(Thiazol-5-yl)dipyrromethane (2).

In a foil-covered 250-mL three-necked flask, equipped with a magnetic stirrer and N, inlet, was placed 5-thiazolecarboxaldehyde (1. 0.88 g, 7.81 mmol) (Dondoni, A.;
Fantin, G.; Fogagnolo, M.; iVledici, A.; Pedrini, P. Syntliesis 1987, 998-1001), CH1CI2 (30 mL), and pyrrole (6 mL, 87 mmol). The reaction mixture was stir*ed for 10 min, then TFA (0.25 mL., 3.2 mrnoI) was added. After a stirring period of 2 h at room temperature, the reaction mixture was transferred to a separatory funnel and washed with saturated aqueous NaHCO3 (50 mL), H,O (50 mL) and brine (50 mL).
The organic laver was dried (Na,SO1), filtered, and concentrated in vacuo. The residue was dissolved in CH,CI_ (50 tnL) and adsorbed onto silica gel (3 g).
Purification by column chromatography (Qradient elution 33-67% EtOAc,'hexanes) provided dipyrromethane 2 (0.95 as a Qrav solid: 'H v',IvIR (300 'VIHz.
CDC13) a 5.74 (s, 1 H), 6.02 (m. 2 H). 6.17 (m. 2 H). 6.70 (m, 2 H), 7.58 (s.
I H), 8.19 (br s, 2 H), 8.68 (s, I H).

2. 5,15-Bis(4-carbomethosvphenyi)-10,10-(thiazol-5-yl)porphyrin (4).
In a foil-covered 250-mL three-necked round bottom flask, equipped with a magnetic stirrer and a N, outlet, was added methvl 4-formvlbenzoate (3, 180 m;, 1.09 mmol), dipvrromethane 2(249 mV. 1.09 mmol), and CH,CI: (110 mL). The reaction mixture was stirred for 15 min. then TFA (0.25 mL, 3.25 mmoI) was added. After a stirring period of 2.5 h at room temperature, DDQ (372 mg, 1.64 nunoI) was added. The reaction mixture was stirred overnight and the solvent was removed in vacuo. The crude residue was adsorbed onto silica gel (3 g) then purified by column chromatography (gradient elution 0-1.5 ro ivleOH/CH,CI,) to provide porphyrin 4 (80 mg, 10 'o yield) as a purple solid: 'H NNIR (300 'vIHz, CDC13) 6 -2.75 (s, 2 H), 4.11 (s, 6 H), 8.28 (d, 4 H), 8.47 (d, 4 H), 8.65 (s, 2 H), 8.82 (d, 4 H), 8.99 (d, 4 H), 9.33 (s, 2 H).

is 3. [5,15-Bis(4-carbomethoryphenyi)-10,20-(thirzol-5-ynporphyrinato]-manganese(III) Chloride (5).
A solution of porphyrin 4(75 mg, 0.101 mmol) and MnC12 (129 mg, 1.03 mmol) in DMF (15 mL) was heated at 125 C for 14.5 h. The mixture was cooled to room temperature while exposed to a stream of air, then concentrated in vacuo.
Repeated chromatographic purification of the product (gradient elution 5-10%
MeOH/CHZCIZ) provided porphryin 5 (7 mg, $%) as a dark green solid: mp >300 C; UV-vis 466.0 nm, E= 1.34 x 10s Llcm-mol; API MS mlz = 797 [C4zHi6MnNbOaSI=

II. [5,10,15,20-Tetrakis(thiazol-5-yi)porphyrmatoJmanganese(IIII) Chloride (7) and [5,10,15,20-Tetrakis(3-methylthiazoiinar5-ynporphyrinatoj-manganese(III) Pentachloride (9).

oxc ~s + x H
~
Propionic Acd a t~-l s /
vs v\ J
I~ s \ ~ \1 MnCI,. DtitF I CH;I. DMF. a Counterion Exchange s N=~

cr S 4cr Ntn` i S / ~IH 2Y

SJ N S'J
\ ~ \
S \
~---v \ /NaOH. ~InCI,S HCI L~-$ 3. Counterion Ecchange cr 8 S v ` N =' / Ivin ~ - / ~
Nr Y SJ
\ ~ \1 S

1. 5,10,15,20-Tetrakis(thiazol-5-yl)porphyrin (6).
A 250-mL three-necked flask equipped with a condenser and chargaed with propionic acid (60 tnL) was heated to reflux. 5-Thiazolecarboxaldehyde (1, 373 mg, 3.30 mmoI), pyrrole (230 L, 3.32 mmol), and an additional 5 mL of propionic acid were added. After 3.5 h at reflu.Y, the mi.rnue was cooled to room temperature while exposed to a stream of air. The solvent was removed in vacuo, the residue was redissolved in CHC13/MeOH/concentratedNIH,OH (6:3:1; 100 mL), and the solvent was removed in vacuo. The residue was adsorbed onto silica ge1 (3 g) and purified by column chromatography (gradient elution, 1-2% '.VIeOH/CH,CI,) to provide porphyrin 6 (123 mQ. 14 ,'0) as a solid: 'H NMR (300 LIHz. CDCI, j 6-2.70 (s, 2 H), 8.67 (s. 4 H), 9.02 (s. 8 H), 9.338(s. 4 H).

2. [5,10,15,20-Tetral:is(thiazol-5-yl)porphyrinato]manganese(III) Chloride (7).
A solution of porphyrin 6(61 m(z. 0.115 mmol) and NInCI, (144 mg, 1.14 mmol) in DNIF (15 mL) was heated at 125 C for 7.5 h. A stream of air was introduced and the reaction mixttue was warmed to I30 C. After a stirring period of 1.5 h, the reaction mixture was cooled to room temperature. The solvent was evaporated in vaczco, and the residue was adsorbed onto silica gel (2 a). Purification by column chromato.graphy (gradient elution. 10-20 % N-,IeOH/CH,CI_) provided porphyrin (36 mg, 43%) as a dark green solid: mp >300 C; UV-vis )~.,,,u = 466.5 nm, E=
3.55 x 10' L/cm-mol; FAB ilviS mr= = 695 [C3zH16'InNgS3]-.

3. 5,10,15,20-Tetrakis(3-methylthiazolium-5-yl)porphyrin Tetrachloride (8).
A solution of 6(12: mg, 0.19 mmol), CH;I (5 mL), and DMF (5 mL) in a sealed tube was heated at 100 C for 2=1 h. The crude porphyrin iodide salt that precipitated out of the reaction mixture was filtered, washed alternately with CH,C12 and ether, and dried under vacuum at room temperature. The iodide was dissolved in water, precipitated out as the he:tafluorophosphate salt (by dropwise addition of aqueous ~1H,`PF6' solution; 1g/I O tnL), filtered, washed with water and isopropanol, and vacuum dried at room temperature. The hexafluorophosphate salt was dissolved in acetone then filtered (to remove insoluble solids). Tne product was precipitated out as the chloride salt from the filtrate by dropwise addition of a solution of Bu4NH;-Cl' in acetone (1 8/I O mL), filtered, washed with copious quantities of acetone, and dried under vacuum at room temperature, to provide porphyrin 8(66 ma, 41%): 'H w1R (300 '.\,IHz. DivISO-do) -3.1 (s, 2 H), 4.6 (s, 12 H),9.49(s,4H),9.38(s,8H),10.85(s,4 H).

4. (5,10,15,20-Tetrakis(3-methvlthiazolium-5-vl)porphyrinato]manganese(III) PentachIoride (9).

Porphyrin 8 (60 ms, mmol) was dissolved in water (1 _5 mL) and the solution pH
was adjusted to pH = 12 by dropwise addition of 6ti' 'NaOH. Solid vfnCl, (I-17 m;) was added into the reaction mixture (the resultin- pH = 8.7). After a stirring period of 30-60 min, the reaction mixture was filtered throua.h a fritted fu.nnel lined with a filter paper. The pH of the filtrate was adjusted to pH = 4-5 (IN HCI) then the solution was filtered. Purification by the double precipitation method (as described for the preparation of 8) provided porphyrin 9 (6 mg, 8.2%) as a dark brown solid: mp >300 C; UV-vis km,X = 460.0 nm. E=
1.25 x lOs L/cm-mol.

III. 15,15-Bis(thiazol-5-yl)porphyrinato]manganese(III) Chloride (12).

s 1. TFA (3 eq) / \ + / CH~CI: NH N
OHC NH H, I Z DpQ H H
HN
1 10 \ \ \ , S \
~_ 1QnC1,, 11 DMF. J
s CI' /N
H H
ti \ \ ~ ~

1. 5,15-Bis(thiazol-5-yl)porphyrin (11).

In a foil-covered 500-mL three-necked round bottom flask, equipped with magnetic stirrer and a N2 inlet, was added dipyrromethane 10 (288 mg, 1.97 mmol) (Chong, R; Clezy, P. S.; Liepa, A. J.; Nichol, A. W. Aust. J. Chem. 1969, 22, 229), 5-thiazolecarboxaldehyde (1, 223 mg, 1.97 mmol), CH,C12 (198 mL) and sodium chloride (13 mg, 0.2 mmol). The reaction misture was stirred vigorously for 10 min, then TFA (0.46 mL, 5.97 mmol) was added. After a stirring period of 40 min, DDQ (671 mg, 2.96 mmol) was added, and the reaction mixture was stirred for an 2;

additional 4 h. The solvent was evaporated in vacuo, and the residue was adsorbed onto silica ;el (3 g). Repeaied chromatographic purification (gradient elution 0.5-2% LfeOH/CH2,Cl2) provided porphyrin 11 (28 mg, 6%) as a solid: 'H 1VMR (300 NIHz, CDC13) &-3.07 (s, 2 H), 8.69 (s, 2 H), 9.21 (d, 4 H), 9.39 (s, 2 H), 9.43 (d, 4 H), 10.35 (s, 2 H).

2. [5,15-Bis(thiazol-5-yi)porphyrinatolmang3nese(III) Chloride (12).

A solution of porphyrin 11 (28 mg, 0.0587 mmol) and VInCI: (85 mg, 0.675 mmol) in DMF (8 mL) was heated at 125 C for 15 h. The mixture was cooled to room temperature while exposed to a stream of air, and the solvent was removed by rotary evaporation. The residue was dissolved in 10 '0 '.IvfeOH/CH,Cl, (50 mL) and adsorbed onto silica gel (500 mg). Purification by column chromatography (gradient of 5-10% ;~feOH/CH,CI,) provided porphyrin 12 (29 mg, 860'0) as a dark brown solid: mp >300 C; UV-vis 457.5 nm. 6 = 3.75 x 10' L/cm-mol; API
NfS m/_ = 529 [C,6H,,tilruv6SJ,'.

IV. [5,15-$is(4carbomethoayphenyi)-10,20-bis(3-methylthiazolium-2-yl)porphyrinato]manganese(III) Trichloride (16).
Co,_Me Nn 1.TF:1.
NaCi.
O CH=Ct=
TFA, CH_CLt ~ ~

S H NH Md ~ Z. DDQ

CO-Me N NH N g S V HyJJJ N
CO. `tt 1. 'vinCl,, DNIF..%
2. CH3I. 45 C
3. Counterion Exchange CO-Nte 3 Cl' CN ~ N~ _N S
~ Ntn S *1 N N-\

CO-Mt f(o 1. meso-(ThiazoI-2-yl)dipyrromethane (14).
In a foil-covered 250-mL three-necked flask, equipped with a magnetic stirrer and a N, inlet, was placed 2-thiazolecarboxaldehyde (13, 0.97 a, 8.6 mmol) (Dondoni, A.;
Fantin, G.; Fogagnolo, M.; Medici, A.; Pedrini, P. Synthesis 1987, 998- I001), CH,C12 (35 mL), and pyrrole (7.2 mL, 104 mmol). The reaction mixture was stirred for 10 min, then TFA (0.26 mL, 3.4 mmol) was added. After a stirring period of I h at room temperature, the reaction mi.Yture was transferred to a separatory ftmnel and washed with saturated aqueous NaHCO3 (50 mL), H,0 (50 mL), and brine (50 mL).
The organic layer was dried (Na:SO,), filtered, and concentrated in vacuo. The residue was dissolved in CH,CI: (50 mL), and adsorbed onto silica gel (3 a).
Purification by column chromatography (1:1 ether;hexanes) provided dipyrromethane 14 (1.22 õ62%) as a solid: `H VNIR (300 MHz. CDCI;) 5 5.78 (s, 1 H). 6.04 (s, 2 H), 6.15 (m. 2 H), 6.71 (m. 2 H), 7.20 (d. 1 H). 7.74 (d. I
H), 8.81 (br s, 1 H).

2. 5,15-Bis(4-carbomethoxvphenyl)-10,20-(thiazol-2-vl)porphvrin (15).
In a foil-covered 1;00-mL three-necked round bottom flask, equipped with a masnetic stirrer and aN, outlet, was added dipyrromethane 14 (771 mg, 3.39 mmol), methyl 4-formvlbenzoate (3, 0.557 g, 3.36 mmol) and CH,CI, (350 mL).
The reaction mixture was stirred for 15 min, then TFA (0.8 mL, 10.4 mmol) was added. After'a stirrina period of 3 h at room temperature, DDQ (1.16 g, 1.64 mmol) was added. The reaction mixture was stirred for 2 d, then the solvent was removed in vacuo. The residue was adsorbed onto silica gel (4 v) and purified by column chromatography (gradient elution 0.5-1% MeOH/CH,CI,) to provide porphyrin 1:5 (140 mg, 11%) as a purple solid: (300 MHz, CDC13) 5 -2.29 (s, 2 H), 4.12 (s, 6 H), 8.02(d,2H),8.30(d,4H),8.44(d,2H),8.47(d,4H),8.84 (d,4H),9.05(d,4H).

3. [:5,15-Bis(4-csrbomethosyphenvl)-10,20-bis(3-methvlthiazolium-2-yl)-porphyrinatojmanganese(III) Trichloride (16).

A solution of porphyrin 15 (26 mg, 0.054 mmol) and "vinC1, (40 ma, 0.40 mmol) in DMF (20 mL) was heated at 135 C ovemight. The s.i.~*,ire was cooled to 45 C
and CH3I (0.8 mL, 11.2 mmol) was added. The reaction mixture was stirred for h at 45 C and DMF was evaporated in vacuo. The residue was purified by colutnn chromatography (gra(iient elution EtOAc. CHC13, MeOH, concentrated NHlOH) to provide the product contaminated with impurities. Following a second purification by column chromatography (6:3:1 CHC13,'MeOH;'concentrated vH10H) non-polar fractions were collected leaving the bulk of product at the top of the column.
The top silica ael containinc, the product was collected and washed with CHCI;/lVfeOH/1N HC1(6:-1:1). Evaporation of the acidic solution provided the product that contained excess inorQanic salts. Purification bv the double precipitation method and vacuum dryinc, at 35 C for 2 d provided porphyrin 16 (9 mg, ?8 .%) as a black solid: mp >300 C; LV-vis =459.0 nm; E= 1.36 x 103 L/cm-mol; API ZMS ml: = 886 [C,,H;,MnN601S,~CHCO."] =.

V. [5,15-Bis(3-methvlthiazolium-2-yl)porphyrinatoJmanganese(III) Trichloride (19).

/ N
NH HN
\S~CHO + C

1. TFA, CHZC12 ? DDQ

N S
NH N
H H l. CH3I. DMF, _V Hti' A. sealed tube \ \ \ ~ \2. Counterion Exchange S ~N ~
-N s 2Cl' IT / / ~ \
/ NH N
H \ H
_`1 1-lli \ \ \ \
Sf ~N \ S 3 C1' 1. NaOH, :~inCl, 2. HCl ~ - S
3. Counterion Exchange H NMaN H
N
\ \ \
S N'--1. 5,15-Bis(thiazol-2-yi)porphyrin (17).
In a foil-covered 500-mL three-necked round bottom flask, equipped with magnetic stirrer and a NZ inlet, was added dipyrromethane (10, 677 mg, 4.6 mmol), 2-thiazol-carboxaldehyde (13, 524 mg, 4.6 mmol), and CA,Cl, (450 mL). The reaction mixture was stirred for 10 min, then TFA (1 mL,16.9 mmol) was added. After a stirring period of I h, DDQ (1.58 g, 7 mmol) was added and the reaction mixture was stirred overni?ht. The solvent was evaporated in vacuo, and the residue was adsorbed onto silica gel (3 a). Repeated purification bv column chromatoqraphv (gradient elution 1-22% MeOH,'CH,CI,) provided porphvrin 17 (51 mv. 4.62%) as a purple solid: 'H NNIR (300 NIHz, CDC13) a -3.05 (s, 2 H), 8.06 (d. 2 H), 8.51 (d, 2 H), 9.35 (d. 4 H), 9.41; (d, 4 H),

10.40 (s, 2 H).

2. 5,15-Bis(3-methyithiazolium-2-yl)porphyrin Dichloride (18).
A solution of porphyrin 17 (140 ma, 0.29 mmol), CH;I (4 mL), and DNIF (20 mL) in a sealed tube was heated at 100 C for 48 h. The precipitate that formed durin;
the reaction was filtered and washed with ether. Purification of the solid precipitate by the double precipitation method provided porphyrin 18 (120 mQ, 71%) as a purple solid: `H NilyIR (300 N,IHz, DMSO-db) 8 -3.4 (s, 2 H), 4.09, 4.06 (2 s, 6 H.
atropisomer INi-CH3), 9.07 (d, 2 H), 9.2 (d, 2 H), 9.4 (d. 4 H), 9.9 (d, 4 H), 10.96 (s, 2H).

3. [:5,15-Bis(3-methylthiazolium-2-yl)porphyrinatolmanganese(III) Trichloride (19).

Porphyrin 18 (120 mg, 0.2lmmol) was dissolved in water (25 mL) and the solution pH was adjusted to pH = 12 by dropwise addition of 6N NaOH. Solid ti1nCl, (147 mg) was added (the resulting pH = 8.7) and the reaction mixture was stirred for 30-60 min. The reaction mixture was filtered through a fritted funnel lined with a filter paper. The pH of the filtrate was adjusted to pH = 4-5 (IN HC1) and the solution was filtered. The filtraze was subjected to the double precipitation method to provide a mixture of porphyrins 18 and 19. The resulting mixture was separated by column chromatography (9:0.5:0.5 CH,CNlwater/saturated I{NO3) to provide porphyrin 19 (25 mg, 18%) as a dark solid: mp >300 C; UV-vis 450.5 nrn, s = 5.99 x 10 L/cm-mol.

VI. [5,10,15,20-Tetraids(1-methylimidazol-2-yl)porphyrinatojmanganese(III) Chloride (22) and [5,10,15,20-Tetrakis(1,3-dimethylimidazolium-2-yl)-porphyrinatojmanganese(III) Pentachloride (24) ~

CHO + H
2.0 Prapionic Acid ~
NN--, CNH NN N Hti N

\ \ \` /
MnCI,. DMF

v NH3L Dh[F. A
N N--cr 2ti ~Nv~- 41-/
N N N
C ~Mn"
N
y '.vH N N' N N N C I
\ \ \ \ / N' _N HN v ~N N
/MnC1,NaOH HCI
cr 3. Counterion Exchange z3 N+ NiMnN
~
\ \
N N`-U
z~}

~1 1. 5,10,15,20-Tetrakis(1-methyiimidazol-2-y4porphyrin (21).
In a foil-covered 1-L three-neck flask equipped with magnetic stirrer, thermometer, and condenser was placed aldehvde 20 (2.0õ 18-1 tnmol) and propionic acid (400 mL). The reaction mhcture was heated to 120 C at which time pyrrole (1.26 mL, 18.2 mmol) was addecL The reaction miYtnre was heated under reflux for an additional 4.5 h and was stirred at room temperature for 3 d. The propionic acid was removed in vacuo, the dark residue was dissolved in a solution of 5%
MeOH%CHZCI, and adsorbed onto silica gel (18 g). Repeated column chromatographic purification provided porphyrin 21 (280 mg, 10%) as a purple solid: 'H NMR (300 iviHz, CDCI;) 5 -2.94, -2.90, -2.84 (3 s, 2 H. atropisomer NH).
3.39-3.58 (multiple s, 12 H. atropisomer N-CH;), 7.50 (d. 4 H), 7.71 (d. 4 H).
8.9=
(m, 8 H).

2. [5,10,15,20-Tetral:is(1-methyiimidazol-2-yl)porphyrinato]manganese(III) Chloride (22).

A solution of porphyrin 21 (29.9 m(z. 0.047 mmol) and MnCI_ (61 mz, 0.48 mmol) in DNIF (12 mL) was heated at 120'C for 14 h. The mixture was cooled to room temperature while exposed to a stream of air. and concentrated by rotary evaporation. Purification by column chromatography (CHCI;/IvfeOH/concenuated NH4OH/EtOAc) provided porphyrin 22 (12.5 mg, 37%) as a black solid: mp >300 C; UV-vis n.mam = 463.0 nm; s = 9.35 x 10' Llcm-mol; API MS m/_ = 68:
[C3eH_sMnN,aF=

3. 5,10,15,20-Tetrakis(1,3-dimethvlimidazolium-2-yl)porphyrin Tetrachloride (23).

A solution of porphyrin 21 (589 mg. 0.934 mmol) and CH3I (3 mL, 48 mmol) in DMF (10 mL) was heated in a sealed tube at 100 C for 14 h. The reaction mixture was poured into ethyl acetate (200 mL) causing porphyrin 23 to precipitate as the iodide salt. The solution was filtered and the brown solid was washed with EtOAc and ether. The product was purified by column chromatography (CH,CN/water/saturated I{.VO) and subjected to the double precipitation method to provide porphyrin 23 (540 mg, 69%) as a purple solid: 'H NMR (300 'v1Hz, DMSO-db) 8 -3.22 (s, 2 H), 3.78 (s, 24 H), 8.60 (s. 8 H), 9.44 (s, 8 H).

4. [5,10,15,20-Tetrakis(1,3-dimethylimidazolium-2-yl)porphyrinatoJmanganese(III) Pentachloride (24).

Porphvrin 23 (1.0 g, 0.33 mmol) was dissolved in 'vIeOH (550 mL) then MnCl, (1.57 g, 12.5 mmol) was added. The solution pH was adjusted to pH = 7.3 , with 6N, NaOH while bubbling a stream of air into the reaction mixture. The pH of thz solution was maintained pH > 7.3 for 1 h then adjusted to pH =-1.5 with IN
HCI.
The solution was filtered and the precipitate subjected to the double precipitation method and dried to provide porphvrin 24 (0.570 g. 74%) as a solid: mp >300 C;
W-vis a,,,,u = 460.5 run; s= 8.38 x 10; L,'cm-mol; FAB NMS m/_ = 778 [C.oHioNIn1N12]-4.

VII. [5,15-Bis(4-carbometho!cyphenvl)-10,20-bis(1-methylimidazol-2-vl)-porphyrinato]man;anese(III) Chloride (27) and [5,15-Bis(-1-carbomethoxy-phenyl)-10,20-bis(1,3-dimethylimidazolium-2-yl)porphyrinato]manganese(III) Trichloride (29).

.,, ~~

f CO1Ms N

+
\ ~~ /

Qi0 I I.1FA, CFi2C1_ 2. DDQ
CO;.tile I\
CN / H N
N
N v ~ N J
fL~i CO_.,Ae ~ \ \ \ `
CO_Me \axchanc CH;I. DMF I
CI' Counterion :' i 2 CI ' /~MF~ C1 CO_Nie N 2r: U- NH
N Q N `

N ( ~.; CO_Me \ \ ~
CO.,Me ~ r /MnCl., DMF.
3 ci CO_Me 2r , , :t = ` N
tiln-N ~
N.
C0.Me 1. 5,15-Bis(4-carbo methozyp henvI)-10,20-b is(1-methylimidazol-2-Yl)porPhyrin (26).

In a foil-covered 500-mL three-necke3 flask, equipped with a magnetic stirrer and N, inlet, was placed dipyrromethane 25 (0.71 g, 3.09 tnrnol), CH,CI, (310 mL), aldehyde 4 (50 mg, 3.09 tnmol), and vaCl (22.4 mg, 0.35 mmol). The reaction mi.~cture was stirred for 10 min, then TFA (1.48 mL, 19.2 mmol) was added.
After a stirring period of 4 h at room temperature, DDQ (1.05 g, 4.65 mmol) was added.
The reaction mixture was stirred overnight and the solvent was removed in vacuo.
The residue was adsorbed onto silica ?el (10 g) then purified by column chromatography (gradient elution'2-4 =b EtOAc: hesanes) to provide porphyrin (220 g 249'o) as a purple solid: 'H ti-MR (300 MHz. CDCI;) 5 -2.85 (s, 2 H), 3.43.
3.49 (2 s, 6 H, atropisomer v-CH;). 4.14 (s. 6 H). 7.49 (d. ? H), 7.68 (d, 2 H), 8.30 (d, 4 H), 8.48 (d, 4 H), 8.87 (m. 8 H).

2. [5,15-Bis(4-carbomethosyphenvl)-10,30-bis(1-methvlimidazol-2-yl)-porphyrinatoJmanganese(III) Chloride (27).
A solution of porphvrin 26 (50.7 mg, 0.071 mmol) and.MnCI, (88.6 mg, 0.70 mmol) in DMF (20 mL) was heated at 120 C for 14 h. The mixture was cooled to room temperature while exposed to a stream of air, then concentrated by rotary evaporation. Purification by column chromatography (gradient elution 5-10%
MeOI-UCH_Cl_) provided porphyrin 27 (25 mg, 42%) as a black solid: mp >300 'C;
LJV-vis a.,,,ac = 463.0 nm; s= 6.70 x 101 L!cm-mol; F.4B N/1S mL- = 791 [C44H3:M1uN$O4]'.

3. 5,15-Bis(4-carbomethoayphenyl)-10,20-bis(1,3-dimethylimidazolium-2-yi)-porphyrin Dichloride (23).

A solution of porphyrin 26 (80 mg, 0.11 tnmol), DNiF (7 mL) and CH3I (0.150 mL) was stirred at room temperature for 4 h. The solvent was removed by rotary evaporation to give a dark colored residue. The residue was purified by column chromatography (CHCI;/11rleOH/concentrated NH4OH/EtOAc) to provide porphyrin 28 (21 mQ, 18%) as a purple solid: 'H NVIR (300 NiHz DNfSO-d5) 5 -3.02 (s, 2 H), 3.73 (s, 12 H), 4.08 (s, 6 H), 8.45 (q, 8 H), 8.56 (s, 4 H), 9.13 (s, 8 H);
API NIS mi=
= 384 [C,6H,,,MruvsOl]-'.

4. [5,15-Bis(4-carbomethosvphenvl)-10,20-bis(1,3-dimethvlimidazolium-2-vl)-porphyrinatolmanganese(III) Trichloride (29).

A solution of porphyrin 28 (19.5 ma, 0.0221 mmol) and MnCl, (22.4 mg, 0.18 mmol) in DiVIF (5 mL) was heated at 120 C for 14 h. The reaction mixture was cooled to room temperature while exposed to a stream of air, then concentrated by rotary evaporation. Purification by column chromatography (CHCI;,~MeOH/concentrated NH3OH/EtOAc) provided crude porphyrin 28.
Purification by the double precipitation method and drying provided porphyrin (6.5 mg, 37%) as a solid: mp >300 C; G"V-vis k,,,,x ==147.5 nm; s= 1.27 x 10' L/cm-mol; FAB MS m/z = 856 [C.,,H,$vlnNsO,] =.

VIII. [5,15-Bis(carboethozy)-10,20-bis(1-methyiimidazol-2-yi)porphyrinatoJ-nnanganese(IIl) Chloride (32).

COZEt N N_~ 0 1. TFA, NaCI, ~ / NH N\
CNrIH4 ~ + ~OEt C~i: rN ND HN ~ O 2. DDQ `N _.~t KY \ N
\ ~ \
;z 5 Co'Et 3l CO-Ei /nC1~ - C~ b1F, a N y N
~~-tn 31 N N I N N
1 \ ~ \~
C0_Et .....

1. 5,15-Bis(carboethory)-10,20-bis(1-methylimidazot-2-yt)porphyrin (31).

In a foil-covered 500-mL three-necked flask, equipped with a magnetic stirrer and NZ inlet, was placed dipyrromethane 25 (0.5 g, 2.2 mmol), CH_Cl2 (220 mL), and aldehyde 30 (225 mg, 2:2 minol). The reaction mhcture was stirred for 10 min, then TFA (1.0 mL, 12.9 mmol) was added. After a stirring period of 2 h at room temperature, DDQ (750 mg, 3.3 mmol) was added, and the reaction mixture was stirred overnight. Triethylamine (2.0 mL) was added, the solvent was evaporated in vacuo, and the residue adsorbed onto.silica gel (10 g). Purification by column chromatography (5% EtOH1CHC13) provided porphyrin 31 (86 mg, I3%) as a purple solid: `H NN1R (300 viHz, CDC13) S-3.08, -3.06 (2 s, 2 H, atropisomer NH), 1.82 (t, 6 H), 3.40, 3.49 (2 s, 6 H, atropisomer N-CH,), 5.11 (q, 4 H), 7.53 (d, 2H),7.72(d,2H),8.94(m,4H),9.50(d,4H).
2. [5,15-Bis(carboethoay)-10,20-bis(1-methylimidazol-2-y!)porphyrinatol-manganese(III) Chloride (32).

A solution of porphyrin 31 (27.7 ms, 0.045 mmol) and vInC12 (59.1 mQ, 0.47 mmol) in DiNtt (12.5 mL) was heated at 120'C for 14 h. Additional MnCI, (29 mg, 0.23 mmol) was added and the reaction mixture was heated for another 2 h.
The reaction mixture was cooled to room temperature while exposed to a stream of air, then concentrated by rotar;J evaporation. Air was bubbled into a3olution of the product dissolved in ethanol with two drops of IN HCI. The solvent was evaporated in vacuo to aive a dark colored residue. Purification by column chromatographv (aradient zlution 10-30% EtOH,'CHC13) provided porphvrin 32 (6.5 mg, 35%) as a black solid: mp >300 C: Lti'-vis 458.5 nm: s= 6.01 x 10' L,'cm-mol; API iMS m/_ = 667 [C;jH_s1N,fn_~;sOaJ".

IX. [5,15-Bis(1-methvlimidazol-2-yl)porphyrinatojmanaanese(III) Chloride (34) and [5,15-Bis(1,3-dimethvlimidazolium-2-yl)porphyrinato]manganese(III) Trichloride (36).

/

C\N7H /
VCHO + ~ /

.2 e 10 I. TFA CFI.CiZ
J2.DDQ
n N N--lYH Y
H H
\ \ \
CH31. DMF
N. V, ~1nCl:. ~ ~ /=~
Cl' D'vfF.~ N 33 2Cl' H N~ iN
1-f ~ ~ ;~i H ~i \ \ \ 1 H / ` H
_V H~i r -~
~~ ~ /aOH. MnCI2 v 'N
, 2 HCl 3 Cl' 3. Counterion Exchange V v H ,Nfn- H
v V
\

N Y' 3(~

1. 5,15-Bis(1-methylimidazol-2-yi)porphyrin (33).
In a foil-covered I-L three-necked flask, equipped with a mapetic stirrer and N, inlet, was placed dipyrromethane 10 (1.0 g, 6.84 mmol), CH,CI_ (680 mL), and aldehyde 20 (753 m?, 6.84 mmol). The reaction mixture was stirred for 10 min, then TFA (3.1 mL, 40.2 mmol) was added. After a stirring period of 2 h at room temperature, DDQ (2.3 g, 10.1 mmol) was added and the reaction mixture was stirred ovemight. Triethylamine (5.75 mL) was added into the reaction mixture, the solvent was evaporated in vacuo and the residue was adsorbed onto silica ;el (15 g).
Purification by column chromatography (611'0 :VIeOH/CH,C1_) provided porphyrin (0.120 g, 7%) as a purple solid: 'H vti1R (300 '*vIHz CDC13) 5 -3.28 (s. 2 H).
3.45.
3.52 (21 s, 6 H, atropisomer N-CH), 7.53 (d. 2 H). 7.7=1(d, 2 H). 9.07 (m. 4 H). 9.46 (d. 4 H). 10.37 (s. 2 H).

2. [5,15-Bis(1-methylimidazol-2-yl)porphyrinatoJmaneanese(III) Chloride (34).
A solution of porphyrin 33 (50 mQ, 0.106 mmol) and 'v1nCl, (130 ma, 1.4 mmol) in DNIF (20 mL) was heated at 120 C for 14 h. The mixture was cooled to room temperature while exposed to a stream of air, then concentrated by rotary evaporation. Purification by column chromatography (33% NIeOH/CHC13) provided porphyrin 34 (321 ma, 53%) as a black solid: mp >300 C; UV-vis km~c =
454.5 rim; E= 4.98 x 101 L, cm-rnol; API MS rnlc = 523 [C,$H,aMrTNs]-.

3. 5,15-Bis(1,3-dimethylimidazolium-2-yl)porphyrin Dichloride (35).
Porphyrin.33 (95 mQ. 0.20 mmol) was dissolved in Dy1F (15 mL), CH3I (0.5 mL.
8.03 mmol) was added, and the reaction mixture stirred for 48 h. The DilvIF
was evaporated in vacuo and the dark colored residue was purified by column chromatoaraphv (gradient elution 30 ' '.,vIeOH/CH,C1, to 6:4:1 CHC13NMe0E-V1N

HCl) to provide porphyrin 35 (150 mg, 99%) as a puaple solid: `H NTNIR (300 IVIHz, DIVISO-d6) 5 -3.54 (s, 2H), 3.79 (s. 12 I-), 8.55 (s 4 I-1), 9.28 (d, 4 H), 11.00 (s, 2 H).

4. [5,15-$is(1,3-dimethyiimidazolium-2-yi)porphyrinatoJmanganese(III) Trichloride (36).

Porphyrin 35 (150 mg, 0.198 mmol) was dissolved in water (50 mL) and the solution pH was adjusted to pH = 12 with 6Y.NaOH. Manganese chloride (375 mg, 2.98 mmol) was added and the reaction mixture was stirred for 30 min. The solution was filtered on a fine fritted filter funnel. the pH of the filtrate was adjusted to pH = 4 (IN HCI) and the solution was filtered. Purification of the solid filter cake bv the double precipitation method and dr:ina provided porphyrin 36 (25.5 mg, 20%) as a solid: mp >300 C; UV-vis _4=17.5 nm; E= 8.66 x 10' L!cm-mol; API >VIS m/c = 554 [C30H,61VIn_ti3=H] =.

X. [5,10,15,20-Tetrakis(1,4,3-trimethylimidazol-2-yl)porphvrinato]-manganese(III)Chloride (39) and [5,10,1:5,20-Tetrakis(1,3,4,3-tetramethvl-imidazolium-2-yl)porphyrinato]manaanese(III)Pentachloride (41).
~

NN +
H
CHO

Propionic acid ~N
~ ~ 1 \
NH V/
~(ei. DhIF II
l WnCI._ u mevH.
-;NN Vv~
1f 1_ ~`~N CI_ N~ 3$' ~ 11 \
N H Y-L , V \ V HN-- N"~. NIn ~~ -N' Y-11~ V
-v_=Y
VV~
CI-1. NaOH. \,InCI.! ~ \ 35 2. H y~ y~ N~
3. Cuuntenon V~V.~I^-Eiahance N V
\ \ \ /
-N. ui L}1 1. [5,10,15,20-Tetrakis(1,4,5-trimethylimidazol-2-yl)porphvrin (38).
1,4,5-Trimethylimidazole-2-carboxaldehyde (37, 750mg, 5.42 mmol), prepared according to literature procedure (Alcaide, E. et al, Tetrahedron 52:15171-(1996)), was dissolved in propionic acid (120 mL) in a 250 mL three neck round-bottom flask equipped with a thermometer and a condenser. The solution was heated to reflux then pyrrole (0.38 mL, 5.42 mmol) was added. The reaction mixture was heated at reflux for an additional 5 h, then cooled to room temperature while exposed to air overnight. The propionic acid was removed by vacuum, distillation yieldinc, a dark solid residue which was adsorbed onto silica 2el.
Purification by column chromatography (gradient elution 5-109% 'N1eOH, CH,C1,) provided porphyrin 38 as a mixture of atropisomers (108 mg. 10.7%). ' HN'.IVIR
(300 'vIHz, CDC13)6--1.90, -2.35, -2.78 (3-1 s. 2H. atropisomer NH), 2.50 (s.
12 H), 2.57 (s, 12 H), 3.15-3.42 (multiple s. 12 H. atropisomer v-CH;). 8.91 (multiple s.
8 H, atropisomer).

2. [5,10,15,10-Td-al,-(1,4;:-ttmmeihr-IBnidazo1-2 yi)porplrvtnnatolman_2anese(III) Chloride (39).
Porphyrin-38 (40 mg, 0.05 mmol) was dissolved in MeOH (7 mL) in a 25 mL
round-bottom flask equipped with a condenser. ManQanese(II) chloride (101 mg.
0.81 mmol) was added and the reaction miYt=e was heated under reflux for 2 h.
Air was bubbled into the reaction mixture for 20 min then methanol was evaporated in vacuo. Purification of the residue bv column chromatoQraphv provided porphvrin 39 as a black solid (12 mg, 27'0): mp>300 C; LTV-vis Imzx=474.5 nm, E=9.74x10;L/cm-mol; API MS m/_=795[C.,,HuNliuV,_J'.

3. [5,10,15,20-Tetrakis(1,3,4,5-tetramethvlimidazolium-2-yl)porphyrin Tetraiodide (40).

Potphyrin 38 (40 mg, 0.05 mmol) was dissolved in DMF (5 mL) in a sealed tube reactor. Methyl iodide (I mL. 16 mmol) was added and the sealed tube heated at 60 C overnight. Dilution of the reaction miYture with EtOAc (100 tnL) resulted in the precipitation of crude product 40 which was collected by vacuum filtration then purified by coiumn chromatography to provide porphyrin 40 as a dark purple solid (25 mg, 35%): 'H N-NiR (300 i'vIHz DhlSO-d6)6-3?0 (s, 2 H), 2.72 (s. 24 H), 3.58 (s, 24 H), 9.40 (s, 8 H).

4. [5,10,15,20-Tetrakis(1,3,-1,5-tetramethvlimidazoiium-2-voporphyrinato]-manganese(III) Pentachloride (41).

Porphyrin 40 (25 mg, 0.02 mmol) was dissolved in methanol (7 mL) in a:cund-bottomed flask (25 mL). NIanganese(IT) chloride (50 ms, 0.4 mmol) was added and the reaction mianue was heated at 60 C for 6 h. vaOH (2N. 2 drops) was added and the reaction miKture stirred for an additional hour. Tne re2ction mis. LI-e Was through celite a filtered nd washed throu_h -with NleOH. Analvsis of the fi1t.-ate 5,,-UV-vis.specrroscopv indicated that the reac;ion was incomplete. The solvent vias evaporated off and the residue redissolved in lvIeOH (7 mL), thea MnCl. (50 mg, 0.4 mmol) was added and the reaction mixture was heated at 60 C for 3 h. Air was bubbled into the reaction mixture for 20 :nin. The resction mixture was filtered over ceiite and washed vvith.MeOH. Evaporation of the solvents in vacuo provided a brow-n residue. Purification of the product bv the double precipitation method provided porphyrin 41 (10 mg, 5101'0) as a brown solid: mp>300 C; Lti-vis 1,.==E51.5 tun. E=9.29x 101 Ucm-mol.

XI. [5,10,15,20-Tetrakis(-t-methv!-1,2,4-triazo(-3-y!)porphyrinatojmanganese(III) Chloride (44).
_W

~N
N

N~ / / 1 \
N N> + Propionic Acid N H N' NH N N -II
CHO LN HN N-N
\ ~ \

N ~ N
~N N MnCI, N~
cr DMF"
IH
N N
~ N~Mn` N~
N ~N N'N
N N' N=J
ML{

1. 5,10,15,.',0-Tetrakis(4-methvl-1,2,4-triazol-3-yl)porphyrin (43).
4-.NIethyl-1,2,4-triazole :-carboxaldehyde (42, 1.06 a, 9.5 mmol), prepared according to literature procedure (Moderhack, D.; Hoppe-Tichy, T. J. Prakt.
Chem/Chem-Ztg. 1996, 338(2), 169-171), was dissolved in propionic acid (180 mL) in a 250-mL three-neck round bottom flask covered with foil and equipped with a condenser. Tne solution was heated to reflux, and then pyrrole (0.66 mL, 9.5 mmol) was added. The reaction mixture was stirred at reflux for an additional 2.5 h. The reaction was then cooled to room temperature while exposed to air over ?
days. Evaporation of the propionic acid under reduced pressure provided a dark residue which was adsorbed onto silica gel. R-,peated purification by column chromatography (gradient elution. CHCI,, ~IeOH. concentrated NH4OH. EtOAc) provided porphyrin 43 (2119 m;, 14.6%) as a solid mixture of atropiosomers: 'H
NNIR (300 MHz. DNiSO-d6) 8 -3.36. -3.13, -3.09 (3 s, 2 H. atropisomer VH).
3.43-3.64 (multiple s, 12 H, atropisomer N--CH;). 9.03 (broad s, 8 H). 9.20 (s. 4 H).

2. (5,10,15,20-Tetrakis(4-methvl-l,2,4-triazoI-3-yl)porphyrinato1manganese(III) Chloride (44).

Porphyrin 43 (77 mQ. 0.12 mmol) was dissolved in DMF (30 mL) in a 100-mL
round bottom flask equipped with a condenser. Manganese (II) chloride (156 mc,, 1.24 mmol) was added and the reaction was heated at 130 C overnight. The reaction mixture was exposed to a stream of air as it cooled to room temperature.
The porphyrin precipitated out upon the addition of CH,CI, (5-10 mL). The solids were filtered and washed with EtOH and CH,CI, to provide porphyrin 44 (45 mg, 51 .'o) as a brown solid: mp >300 C; UV-vis n~1C =452.5 nm; s= 8.10 x 10' Li cm-mol; FAB-2NIS m/z = 787 [C3zHõi'v 1n:v131 .

XII. [5,15-Bis(trifluoromethyl)-10,20-bis(imidazoi-2-y1)porphyrinatoj-manganese(III) Chloride (47).

N
CF; 1. TFA (6 eN ~ N CH2C1_ C\N?H ~ N
HN ~ CHO 2. DDQ F3C CF3 HN
4!5 = C \ \ \ ~

N N
__N` / V Cl MnCI, `I( DMF-/ / 1 \
N, _.,v F3C / ~ In \ CF3 V
\ \ \

N N
"4 1. 5,15-Bis(trifluoromethyl)-10,20-bis(imidazoI-2-yl)porphvrin (46).
In a foil-covered 1-L three-neck round bottom flask, equipped with a magnetic stirrer and a N2 outlet, was added dipyrromethane 45 (1.13 g, 5.28 mmol), 1-methylimidazole-2-carbosaldehyde (20, 582 mg, 5.28 mmol), sodium chloride (32 mg, 0.54 mmol) and CH2C1, (530 mL). The reaction mi:cture was stirred for 10 min, then TFA (2.40 mL, 31.1 mmol) was added. After a stirring period of 105 DDQ (1.81 g, 7.97 mmol) was added, and the miYture was stirred overnight.
The solvent was removed by rotary evaporation, and the crude residue was adsorbed onto silica ?el (3 g). Purification bv column chromatography (gradient elution, 5-10% T'VfeOH/CH:C1,) provided porphyrin 46 (455 mg, 34%) as a black solid: 'H

NMR(300MHz,CDC13)a-2.87(s,2H),3.56(m,6H),7.85(d,2H),8.05(d,2 H), 8.99 (m, 4 H), 9.81 (m, 4 H); API-MS m/z = 607 [C70H,oF6N8 = H]'.

2. [5,15-Bis(trifluoromethyl)-10,20-bis(imidazol-2-yl)porphyrinatoJmanaanese(III) Chloride (47).

A solution of free porphyrin 46 (113 mg, 0.186 mmol) and vInCI: (360 mg, 2.86 mmol) in DNiF (15 mL) was warmed to 120 C for 6 h. The mixture was cooled to room temperature while exposed to a stream of air, then concentrated by rotary evaporation. The crude residue was dissolved in 1001'0N1eOH/CH:C1, (100 mL), then adsorbed onto silica gel (1 g). Purification by column chromatography (100,%
IVIeOFUCH,CI_) provided porphyrin 47 (45 mg. 359'0) as a dark ?reen solid: mp >300 C; LN-vis 456.5 nm; e = 1.98 x 10{ L'i cm-mol; API-tiIS m/: = 659 [CsoHisFSMtu~Js]'.

XIII. [5,10,15,20-Tetrakis(1-methylpvrazol4-y1)porphyrinatojmanganese(TII) Chloride (50) and [5,10,15,20-Tetrakis(1,2-dimethylpyrazolium--i-yl)porphyrinatoj-manganese(III) Pentachioride (52).

~
N~v / 1 +
CHO H
T 'j P-oplonlc 4xd /
N-N

1. Mei. DhiF. J N VH N
v 2. Counterion M
ni iz.
E.rc.'~ange ~ N HN McOH.
\ / \ \ \
N=v /
N-N
4 C1"
N-N ~ cr /
N 14 cf \

HN N\ Mn`~ ~ = ~ ~~t 1 v v ~
\ \ \ \ \ \1 N-V-/ \ 5 Cr N-N
~- /
~
1. HaOH, ~InC12 V
3. Counteri on v ~fn\y ~\
Excliange \ \ \
N-v-/ \
5z 1. 5,10,15,20-Tetrakis(1-methylpvrazol4-yl)porphyrin (49).

To a refluxing solution of propionic acid (200 mL) and I-methylpvrazole4-carboxaldehyde (48, 0.92 g, 8.32 tnmol), prepared according to Iiterature procedure (Finar, I. L.; Lord, G. H. J. Chem. Soc. 1957, 3314-331 5), was added pyrrole (0.63 mL, 8.32 mmol). The reaction was covered with foil and was heated under reflux for 3.5 h. Upon cooling the reaction mixture was exposed to air overnig.ht.
The propionic acid was then removed by vacuum distiIlation. The crude residue was dissolved in 5% MeOHICH,CI,, then adsorbed onto silica gel (5.3 g).
Purification by column chromatography (5% 1YfeOH/CH,Cl,) provided porphyrin 49 as a purple solid (231 mg, 17.5%): 'H NWR (300 NIIiz. DIVfSO-d6) 5 -2.74 (s, 2 H), 4.28 (s. 12 H), 8.31 (s, 4 H), 8.67 (s, 4 H), 9.16 (s. 8 H).

2. [5,10,15,20-Tetral:is(1-uiethvtpyrazol4-yl)porphvrinatoJmanaanese(III) Chloride (50).

Porphyrin 49 (50 ma, 7.93 x 10'2 mmol) was dissolved in DkIF (10 mL) in a 25-tn.L
round bottom flask equipped with a condenser. Nfanganese (II) caloride (150 ma, 1.19 mmol) was added and the reaction was heated at 125 C for 4 h. A stream of air was introduced and the reaction heated for an additional 2 h. The reaction was diluted with EtOAc (100 mL) and the crude product was collected by vacuum f Itration. Purification of the residue by colurrin chromatography (10%
MeOI-iICH,CI,) followed bv counterion exchange provided porphryin 50 as a?reen solid (15 mg, ?5 '0): mp >300 C; UV-vis a.,o,,= 471.0 run, E= 9.55 x10j Ucm-mol; API MS nx/_ = 683 [C36H_$IvInN,2]-. .

3. 5,10,15,20-Tetrakis(1,2-dimethylpyrazolium-4-yl)porphyrin Tetrachloride (51).

Porphyrin 49 (200 ma, 0.32 tnmoI) was dissolved in DNIF (15 mL) in a sealed tube reactor. Methyl iodide (2 mL, 32 mmol) was added and the sealed tube heated at 125 C for 6 h. Dilution of the reaction mixture with EtOAc resulted in the precipitation of crude product which was collected by vacuum filtration and initially purified by column chromatography (8:1:1 CH3CN/water/saturated K_~tO3).
Further purification by the double precipitation method provided porphyrin 51 as a dark purple solid (45 mg, 17%): 1H titilR (300 vlIiz, DMSO-ds) 6 -3.16 (s, 2 H), 4.55 (s, 24 H), 9.45 (s, 8 H), 9.50 (s, 8 H).

4. [5,10,15,20-Tetrakis(1,2-dimethylpyrazolium4-yl)porphyrinatojmanganese(III) Pentachloride (52).

Porphyrin 51 (=10 mg, 4.80 x 10-2 mmol) was dissolved in u=ater (10 mL).
Manganese (II) chloride (90 mg. 0.72- mmol) was added and the reaction was heated at 50 'C. Analysis of the reaction mixture by L V-vis spectroscopy showed incomplete reaction. Additional NinCl, (210 mg, 1.67 mmol) was added and heatina of the reaction mixture was continued until completion of reaction was indicated by Lti'-vis analysis. Filtration followed by purification of the product by the double precipitation method provided porphyrin 52 (25 m(Z, 57%) as a brown solid: mp >300 C; LV-vis 461.0 nm.

E= 7.82 x 101 L!cm-mol; API NIS nv: = 683 [C10H,oi/ImNi_ -=1CH3]-.

XIV. [5,10,15,20-Tetralos(1,3-dimethyiimidazolium-5-yl)porphyrinatoJmanganese(III) Pentachloride (56).

~~
~N /
_/, N1 `~CHO H

Propioaic acid rN
/ / 1 \ \
'3H N ~I
Nfef, DhiF r-- N \ / ~ \ 1, I
A N `

rv --v/ v 4 r ~,=1 NI N N

LN V ~ \ r-N=1' V
/ 5 Ci"
e ~ / / 1 \
l. NaOH. MnC1: y V iY, 3. Counterion N v~~n~N N\
Exchange / N' W
~
/
a'b 1. 5,10,15,20-Tetrakis(1-methylimidazol-5-yi)porphyrin (54).
To a refluxing solution of propionic acid (400 mL) and 1-methylimidazole-5-carboxaldehyde (53, 2.0 ?, 18.16 mmol), prepared according to literature procedure (Dener, J. M.; Zhang, L-H.; Rapoport, H. J. Org. Chen 1993, 58, 1159-1166), was added pyrrole (1.26 mL, 18.16 mmol). The reaction was covered with foil then heated under reflux for 5 h. Upon cooling, the reaction mixture was exposed to air for 60 h. The propionic acid was then removed by vacuum distillation. The residue was dissolved in 10% MeOH/CH:Cl,, then adsorbed onto silica gel (6 ;).
Purification bv column chromatographv (gradient elution, 5-10% MeOH/CH,Cl,) provided porphyrin 54 as a purple soiid (600 mg, 21%): 'H ~,ti1R (300 N,/IHz.
CDC13) 8-2.80,-2.75 (2 s, 2 H. atropisomer vH). 3.42-3.58 (multiple s, 12 H.
atropisomer N-CH;). 7.87-7.98 (multiple s. 4 H, atropisomer), 8.06 (s. 4 H), 8.9-i-8.99 (multiple s, 8 H, atropisomer).

2. 5,10,15,20-Tetrak-is(1,3-dimethvlimidazolium-5-yl)porphvrin Tetraiodide (55).

Porphyrin 54 (395 mg, 0.63 mmol) was dissolved in DMF (15 mL) in a sealed tube reactor. Methyl iodide (2 mL, 32 mmol) was added and the sealed tube was heated at 100 C overni;ht. Dilution of the reaction mixture with EtOAc (200 mL) resulted in the precipitation of the crude product which was collected by vacuum filtration. Purification by column chromatography (8:1:1 CH3CN/water/saturated K.O3) provided porphyrin 55 (250 ma. 33%) as a dark purple solid: `H NMR (300 MHz, DMSO-d6) 5 -3.25 (s. 2 H), 3.46-3.64 (multiple s, 12 H, atropisomer), 4.30 (s, 12 H), 8.68 (s, 4 H), 9.48 (s. 8 H), 9.78 (s. 4 H).

3. [5,10,15,20-Tetralds(1,3-dimethytimidaaolium-5-yl)porphyrinatoJ-manganese(III) Pentachloride (56).

Porphyrin 55 (200 mg, 0.17 mmol) was dissolved in methanol (100 mL).
Manganese (II) chloride (315 mg, 2.50 mmol) was added and an air stream introduced into the reaction mi.Yture. The pH of the solution was maintained at 8 by the dropwise addition of 6N NaOH over the period of the reaction, after which time the pH was adjusted to 5 with 6N HCI. The reaction was filtered on a fritted funnel.
Purification of the product by the double precipitation method provided porphyrin 56 (63 mg, 41%) as a brown solid: mp >300 C; UV-vis 454.0 nm, s= 1.23 x 103 L/cm-mol.

XV. [5,15-Bis(4-fluorophenyl)-10,20-bis(1-methylimidazol-2-yi)porphyrinatoJ-manganese(III) Chloride (59) and [5,15-$is(4-flnorophenyl)-10,20-bis(1,3-dimethylimidazolium-2-yi)porphyrinatoJ manganese(III) Trichloride (61).

F
N IN N~, C'Nr4H ~~ ~ CHO
1. TFA (6 eq) CH,Ci, 2. DDQ
n N N
/ / 1 \
vH V -1. N[ef. DMF, G F F ~tnCh.
2. Counterion HIN D~tF. J
Exchange 1 \ \ \
N N
2CC Nti N
NH v F F F [n~ F
\ \ \ 1 \ \ \

~N or~
~N N
toc - Y" N-- 3 cr 1. Mn(OAc)z, ivteOH. 55 'C v 2. Counterion F ~(n; F
Erchange - ~( ,( \ \ ~
~~ ~~=~
~
~01 1. 5,15-Bis(4-fluorophenyl)-10,20-bis(1-methylimidazol-2-yl)porphyrin (58).
In a foil-covered 1-L three-neck round bottom flask. equipped with a magnetic stirrer and a N, outlet, was added dipyrromethane 25 (1.00 g, 4.43 mmol), 4-fluoro-benzaldehyde (57, 550 mg, 4.43 mmol), sodium chloride (30 mg, 0.5 mmol) and CH.C1, (450 mL). Tne reaction mixture was stirred for 10 min, then TFA (2.0 mL, 26 mmol) was added. After a stirring period of 105 min, DDQ (1.51 g, 6.65 mmol) was added, and the mixture was stirred overnight. The solvent was removed by rotary evaporation, and the crude residue was adsorbed onto silica cyel (3 a).
Purification by column chromatography (a adient elution, 5-10% N 1eOH/CH,C1,) provided porphyrin 58 (229 mg, 16%) as a black solid: `H NMR (300 NIHz.
DMSO-d,) 6-3.05 (s. 2 H), 3.70, 3.72 (22 s. 6 H. at`opisomer N-CH;). 7.73 (m.

H), 8.19 (s. 2 H), 8.30 (m. 4 H), 9.02 (m. 6 H): A_PI-MS m/_ = 659 [C,,H,gF,Ns ~
H]-.

2. [5,15-Bis(4-fluorophenv1)-10,30-bis(1-methvlimidazol-2-yl)porphyrinato]-manganese(III) Chloride (59).
Porphyrin 58 (85 mg, 0.13 mmol) was dissolved in DNMF (7 mL) in a50-mL round bottom flask equipped with a condenser. lvfanQanese (II) chloride (2715 mg, 1.71, mmol) was added and the reaction was heated at 120'C for 3.5 h. The reaction was cooled to room temperature then concentrated bv rotary evaporation. The crude residue was dissolved in 20 % v1eO1-i/CH_C1, (100 mL) and adsorbed onto silica gel (2 g). Purification by column chromatography (gradient elution, 3-8 'o N-IeOH/
CH:CI2) provided porphryin 59 as a?reen solid (15 mg, 16%): mp >300 C; UV-vis n.m~~ _463.0 nm, E=4.05 x l0y L/cm-mol: :kPI IvIS m/c = 711 [C,oH,6F,NIruvB]-.

3. 5,15-Bis(4-fluorophenyl)-10,120-bis(1,3-imidazolium-2-yl)porphyrin Dichloride (60).

Porphyrin 58 (170 mg, 0.26 mmol) was dissolved in DNMF (7 mL) in a sealed tube reactor. i'viethyl iodide (6 mL, 96 mmol) was added and the sealed tube was heated at 100 C overnight. Tne mixture was cooled to room temperature and concentrated by rotary evaporation. The residue was precipitated as the chloride salt from acetone by the addition of Bu,NCI solution in acetone (0.3 g/mL). The solid was collected on a fritted funnel, washed with copious quantities of acetone, and dried under vacuum at room temperature to provide porphyrin 60 as a dark purple solid (196 mg). T'ne product was used without further puriiication.

4. [:5,15-Bis(4-fluorophenvl)-10,20-bis(1,3-dimethvlimidazolium-2-yl)porphyrinato]manganese(III) Trichloride (61).

Porphyrin 60 (196 mg, est. 0.26 :nmol) dissolved in 'vIzOH (30 mL) was slowly warmed to 55 C then )/In(OAc)3=? H,O (694 m~. 2.59 m.-nol) was added. After a stirring period of 3 h, the mixture was cooled to room temperature. filtered throush Celite and concentrated by rotari evaporation. The residue was purified bv the double precipitation method to provide porphvrin 61 (102 mg, 46% over two steps) as a dark green solid: mp >300 'C, L`v'-vis nma~ =453.0 tun; E = 1.30 x 10' L.'cm-mol; ES-MS m/-= 967 ((Ct2H52F,1,iruti3)-3 T Z(CF;CO_')]-.

XVI. [5,10,15,20-Tetralds(1,3-diethyiimidazolium-2-yi)porphyrinato]manganese(III) Pentachloride (65).

!--\
\,NN + N
y H
CHO

~z Propionic Hid ~Y Y

CN
Ecl. DMF
N __N ELN N

N - N Nv a r ,( NH N N
- . ) N -N ELN iv /~ N -' = N'~
U ~~n'/
ci-/
.i l. Nin(OAc)3. A N ~ N N
2. Counterion C - ,Mn-Exchange -v 11 N N
~ \ \ \

1. 5,10,15,20-Tetrakis(1-ethylimidazol-2-yi)porphyrin (63).

To a refluxing solution of propionic acid (450 mL) and 1-ethylimidazole-2-carboxaldehyde (62, 2.5 g, 20.0 mmol, prepared in a similar manner as the methyl imidazole derivative 20) was added pyrrole (1.40 mL, 20.0 mmol). The reaction was covered in foil then heated under reflux for 5 h. Upon cooling, the reaction mixture was exposed to air overnight. The propionic acid was then removed by vacuum distillation. Repeated purification by column chromatography (gradient elution, CHCl3/MeOHI concentrated V"H,OH/EtOAc) provided porphyrin 63 as a purple solid (281 ms, 8.1%): 'H N,,MR (300 I~IHz. CDCl3) 6 =2.96.-2.90, -2.87 (3 S.
2 H, atropisomer~iH), 0.85-1.26 (multiple t. 1'-'H. atropisomer CH;), 3.61-3.88 (multiple q, 8 H. atropisomer CH,). 7.55 (d. 4 H), 7.70 (d. 4 H). 8.98 (multiple s. 8 H, atropisomer).

2. 5,10,1:5,20-Tetral:is(1,3-diethylimidazolium-2-vl)porphvrin Tetraiodide (64).
Porphyrin 63 (106 ma, 0.15 mmol) was dissolved in D-'-vIF (5 mL) in a sealed tube reactor. Ethyl iodide (10 mL, 25 mmol) was added and the sealed tube was heated at 65 C for 6 h. Dilution of the reaction mixture with EtOAc (100 mL) resulted in the precipitation of the crude product which was collected by vacuum f ltration, washed with chloroform and then purified by column chromatography (8:1:1 CH3Ci`i/water,'saturated K.~1O3) to provide porphyrin 63 (140 m?, 69%) as a dark purple solid_ 'H NI'MR (300 vlHz, D:v1S0-cl,) 3 -3.22 (s. 2 H), 1.17 (t. 24 H). 4.01 (s, 16 H), 8.70 (s, 8 H), 9.43 (s, 8 H).

3. (:5,10,15,20-Tetrakis(1,3-diethylimidazolium-2-vl)porphyrin3toj-manganese(III) Pentachloride (65).

Porphyrin 64 (106 ma, 8.09 x 10' mmol) was dissolved in methanol (1 -; mL) then Mn(OAc)3=2 H,0 (216 mg, 0.81 mmol) was added and the reaction heated at 55 C

for 2.5 h. The reaction was filtered through celite and then evaporated in vacuo.
Purification of the product by the double precipitation method provided porphyrin 65 (65 mg, 78%) as a brown solid: mp >300 C, UV-vis 1bõx = 446.5 nm, E= 1.35 x 10s L/cm-mol; ES-MS m/z = 1307 [(C4ji,6MnNjs+ 4(CF3CO2-)]'.

XVII. [5,10,15,20-Tetralds(1-ethyl-3-methylimidazolium-2-yl)porphyrinato]-manganese(III) Pentachloride (67).

~,N ~,N N-_/ 4C

CN / NH :-<: . , CMnD
N _2. Bu4NC1 in N --N N N
acetone \ \ ~ ~ 1 ( N N
~VN
~- ~ 1. Mln(OAc), MeOH, >j 'C
2. Counterion Exchange --tv vI/
s ci-I
N N N
C ~ / Mn ~
N --N ~ N N
\ \ ~~ [
~NLN

1. 5,10,15,20-Tetrakis(1-ethyl-3-methylimidazolium-5-yl)porphyrin Tetrachloride (66).
Porphyrin 21(371 ma, 0.588 mmol) was dissolved in DMF (8 mL) in a sealed tube reactor. Ethyl iodide (7 mL, 88 mmol) was added and the sealed tube was heated at 60 C overnight. The mixture was cooled to room temperature and concentrated by rotary evaporation. The residue was dissolved in water (20 mL) and purified by the double precipitation method to provide porphyrin 66 (349 mõ 67%) as a dark purple solid: 'H NMR (300 MHz, DMSO-d6) 5 -3.23 (s, 2 H), 1.17 (m, 12 H), 3.77 (m, 12 H), 4.03 (m, 8 H), 7.01, 7.18, 7.35 (multiple s, 8 H), 8.63 (d, 4 H), 9.36 (s, 4 H).

2. [5,10,15,20-Tetrakis(1-ethyl-3-methylimidazolium-2-yl)porphyrinato]-manganese(III) Pentachloride (67).
Porphyrin 66 (340 mg, 0.39 mmol) was dissolved in methanol (45 mL) then Mn(OAc)3=2 H20 (680 mg, 2.53 mmol) was added, and the mixture was stirred at 55 C for 3.5 h. The mixture was cooled to room temperature, filtered through Celite (to remove insoluble solids), and concentrated by rotary evaporation.
The residue was purified by the double precipitation method to provide porphyrin (324 mg, 85%) as a brown solid: mp >300 C; UV-vis X,,,,,, = 446.5 nm; e =
5.11 x 104 L/cm-mol; ES-MS ni/ = 1251 [(C44H4B'VInNI2)+' + 4(CF3C02_)]+.

Treatment of Bronchopulmonary Dysplasia Using Aeol-V (10123) Neonatal baboons were delivered prematurely by Caesarian section and then treated either with 100% oxygen or only sufficient PRN FIO2 to maintain adequate arterial oxygenation. To establish the model, thirteen 100% oxygen treated animals and seven PRN control animals were studied. Treatment with 100% oxygen results in extensive lung injury manifested by days 9 or 10 of exposure and characterized by delayed alveolarization, lung parenchymal inflammation, and poor oxygenation.
This is characteristic of the human disease, bronchopulmonary dysplasia, and is thought to be mediated, at least in part, by oxidative stress on the developincy neonatal lung. In a first trial of Aeol-V, a neonatal baboon was delivered at days gestation and placed in 100% oxygen. The animal received 0.25 mg/ka/24 hr given i.v. in a continuous infusion over the entire 10 day study period (see Fig. 2).

This animal showed marked improvement of the oxygenation index. There was no evidence of clinical decompensation of the lungs at days 9 and 10. This suggests that Aeol-V can be used to treat oxidant stress in the premature newborn.
* * * * :

One skilled in the art will appreciate from a readinz of this disclosure that various chan-es in form and detail can be made without departinc, from the true scope of the invention.

Claims (29)

WHAT IS CLAIMED IS:

1. A compound of formula or pharmaceutically acceptable salt thereof, wherein R1 and R3 are the same and are:
R2 and R4 are the same and are:

Y is halogen or -CO2X, and X is the same or different and is an alkyl group and each R5 is the same or different and is H or alkyl, wherein when R1 and R3 are -H, R2 and R4 are not when R1 and R3 are -H and R2 and R4 are said compound is complexed with a metal selected from the group consisting of manganese, iron, copper, cobalt and nickel.

2. The compound according to claim 1 wherein R1 and R3 are the same and are:

Y is -F or -CO2X and X is the same or different and is a C1-4 alkyl group and each R5 is the same or different and is H or C1-4 alkyl.

3. The compound according to claim 1 wherein R1, R2, R3 and R4 are

4. The compound according to claim 2 or 3 wherein X is methyl or ethyl.

5. The compound according to any one of claims 1-4 wherein R1, R2, R3 and R4 are the same.

6. The compound according to claim 5 wherein R1, R2, R3 and R4 are

7. The compound according to any one of claims 1-6 wherein said compound is complexed with a metal selected from the group consisting of zinc, iron, nickel, cobalt, copper and manganese.

8. The compound according to claim 7 wherein said metal is manganese.

9. A method of protecting cells from oxidant-induced toxicity comprising contacting said cells ex vivo with a protective amount of a compound of formula or pharmaceutically acceptable salt thereof, wherein R1 and R3 are the same and are:

Y is halogen or -CO2X, and X is the same or different and is an alkyl group and each R5 is the same or different and is H or alkyl, so that said protection is effected.

10. The method according to claim 9 wherein said compound is complexed with a metal selected from the group consisting of manganese, iron, copper, cobalt, nickel and zinc.

11. The method according to claim 10 wherein said metal is manganese.

12. The method according to any one of claims 9-11 wherein said cells are mammalian cells.

13. The method according to claim 12 wherein said cells are cells of an isolated organ.

14. The method according to claim 12 wherein said cells are cells of an organ transplant.

15. A compound of the formula or pharmaceutically acceptable salt thereof, wherein R1 and R3 are the same and are:

Y is halogen or -CO2X, each X is the same or different and is an alkyl group and each R5 is the same or different and is H or alkyl, for use in:
(a) protecting cells from oxidant-induced toxicity;
(b) treating a patient suffering from a condition that result from or that is exacerbated by oxidant-induced toxicity;
(c) treating a pathological condition of a patient resulting from degradation of NO or a biologically active form thereof;
(d) treating a patient for an inflammatory disease; or (e) treating a patient for an ischemic reperfusion injury.

16. The compound according to claim 15, wherein said compound is complexed with a metal selected from the group consisting of manganese, iron, copper, cobalt, nickel and zinc.

17. The compound according to claim 16, wherein said metal is manganese.

18. A compound according to any one of claims 1-8, for use in:
(a) protecting cells from oxidant-induced toxicity;

(b) treating a patient suffering from a condition that result from or that is exacerbated by oxidant-induced toxicity;
(c) treating a pathological condition of a patient resulting from degradation of NO or a biologically active form thereof;
(d) treating a patient for an inflammatory disease; or (e) treating a patient for an ischemic reperfusion injury.

19. The compound according to any one of claims 15-18 wherein said inflammatory disease is an inflammatory lung disease.

20. The compound according to claim 19 wherein said inflammatory lung disease is bronchopulmonary disease, asthma or pulmonary fibrosis.

21. The compound according to any one of claims 15-18 wherein said ischemic reperfusion injury results from a stroke.

22. Use of a compound of the formula or pharmaceutically acceptable salt thereof, wherein R1 and R3 are the same and are:

Y is halogen or -CO2X, each X is the same or different and is an alkyl group and each R5 is the same or different and is H or alkyl, in the preparation of a medicament for (a) protecting cells from oxidant-induced toxicity;
(b) treating a patient suffering from a condition that result from or that is exacerbated by oxidant-induced toxicity;
(c) treating a pathological condition of a patient resulting from degradation of NO or a biologically active form thereof;
(d) treating a patient for an inflammatory disease; or (e) treating a patient for an ischemic reperfusion injury.

23. The use according to claim 22, wherein said compound is complexed with a metal selected from the group consisting of manganese, iron, copper, cobalt, nickel and zinc.

24. The use according to claim 23, wherein said metal is manganese.

25. Use of a compound according to any one of claims 1-8 for :
(a) protecting cells from oxidant-induced toxicity;
(b) treating a patient suffering from a condition that result from or that is exacerbated by oxidant-induced toxicity;
(c) treating a pathological condition of a patient resulting from degradation of NO or a biologically active form thereof;
(d) treating a patient for an inflammatory disease; or (e) treating a patient for an ischemic reperfusion injury.

26. Use of a compound according to any one of claims 1-8 for the manufacture of a medicament for:
(a) protecting cells from oxidant-induced toxicity;

(b) treating a patient suffering from a condition that result from or that is exacerbated by oxidant-induced toxicity;
(c) treating a pathological condition of a patient resulting from degradation of NO or a biologically active form thereof;
(d) treating a patient for an inflammatory disease; or (e) treating a patient for an ischemic reperfusion injury.

27. The use according to any one of claims 22-26 wherein said inflammatory disease is an inflammatory lung disease.

28. The use according to claim 27 wherein said inflammatory lung disease is bronchopulmonary disease, asthma, pulmonary fibrosis.

29. The use according to any one of claims 22-26 wherein said ischemic reperfusion injury results from a stroke.