CA2189528A1 - Methods of use for peroxynitrite decomposition catalysts, pharmaceutical compositions therefor - Google Patents

Abstract

The present invention provides a method for the treatment of diseases by the decomposition of peroxynitrite, preferably decomposition to benign products, comprising the use of a complex which is a selected ligand structure providing a complexed metal such as Mn, Fe Ni and V transition metals. The method of use, as well as novel pharmaceutical compositions therefor, are for the treatment of diseases advantageously affected by decomposition of peroxynitrite at a rate over the natural background rate of decay of peroxynitrite in humans suffering from the disease which comprises administration of an amount of a complex, in dosage unit form, which is effective for such acceleration of the decomposition of peroxynitrite.

Description

woss/3lls7 r~l,o~

~ OF I~SE FOR ~;Ku~RrrE DECO~OSlTION
5 i~.ATAT,Y~pT-TARMAil~lmCALCOMPa~ D 'l~;l~;~ )K
TPf'hnif'Al FiPlfl The present invention i6 for methods of use for the d~ of pclu~,~lliLLiLr~ by metal ~ novel 1 C~ hf~ and methods of use therefor.
1~ raL L~UI~LIY~ the present invention now provides a method for treating 6elected diseases ~ c;..g the flF "~ n of ~c~uAy~lihiLc with the use of 8 0~ V' ~fl which is a metal comples.
This rlFf - ~ u~f~ ly produces benign agents ~.c._ALing fnrrnAhnn Of flF~ dF~ u~ products such as osygen 16 radicals and which also further prcvents - v_Lu.l of Dul~_.u~iv~F
.1;~.. 1~ .~ (SOD) by the presence of p_, u.ylliLL iLc. Therefore, the method of use fûr selected metsl c~ npl ~ of the present invention, as well as novel rkA....~ I u~ c for such use is for the iL~r~iluL.Ii, of diseases avlv~u~ ûuAsly affected by ~-C~hu_,l~ f. - -.l- ;~;,.fr O flF. ~ \V~;I .^~n of ~h_~ uA~yllihiic at a rate "~ over a natural L~ ,-uuuld rate of decay which ~^nrnrlAcPc n l~ of an rate-~. .~1. .AI ~E~ effective amount of the metal comples in unit dosage form.
In other words, the methods of L.cc.~uc. . ~ and novel - 25 ~ :----c of this inYention provide a twofold benefit in the 1..cc.Llucllt of diseases (lj ^~ d rate of catalytic ~ : --. of uc.u~. ihi~e and (2) ~IUL__LiUII of SOD against ill&~Liv~iul- by _, uA~ylliLIiLc~
Thus, the present invention provides for a method of 31 ~L~..hu~ of human diseases adv~ultn~cuusly affected by such dP~ l~h~;i . by l..ùt4..hul~ from the dPlptp~im~c effects resulting from the presence of p~.u~vllitLilc in the human body not ~ ' ^ c known.
In addition, since ~ui,~tiu~ against SOD i l&LLivc.~iu.. is provided, such dr- ~ offers protection against diseases r ~ ' ~ with the 35 v~ J.uduction of DU,u~.U~ id~.
These diseases include ischemic Irl~ r.. iDJuries such as stroke, head trauma and AUyu~vli~l ischemia, sepsis, chronic or acute; ~n^- ~nl: . (such as arthritis and ;~n~ ~ ~l U~ bowel disease and the like), adult l~ tVI,~ distress syndrome, cancer, -wo ss/3ll97 2 1 8 9 5 2 8 Y dy8plaBia, 8ide effects from drug ~ ..t of cancer, ~Luvc.s~ular diseases, diabetes (not included for L.t:~.l,-l..,.-l by v~ul~aliu"- porphyrin ~ ultiple sclero6is, p .1~;....,..'5 disease, familial aLu~uLLu~ , lateral sclerosis, and colitis and specific 5 neuronal disorders, preferably ischemic ~ rl~. . r. . -: .. . infl ~ . " "
sepsis, rnultiple sclersis, ~ .'8 disease and stroke.
Rrrl~ ...fl Art Nitric o~ide (NO) is known for it3 dul pl~ 1 ~, - 1 role as helpful ... ~ r- _nd har~l ' ' - ' ' Nitric o~ide is shown to 10 be generated in UU~,~UUD cell types including ~ .u~ha,~."
neutrophils, lle~uflLu~ s and r..~ liAl cells. &e Hibbs et al, ~, 1987, 235, 4',73-476; Rirnele et al, J. rl.A. ~ .p Th~r 1988, 245, 102-111; Curran et al, J. ~.Yr ~Pfl 1989, 170, 1769-1774; and Pla~ner et al, Nature, 1987, 327, 524-526; I~ ,t .i~ly. rne chemical reaction 15 r~sr~nQ;l '- for the production of NO is catalyzed by a class of enzymes referred to as nitric oxide synthases (NOS) which conYert L-arginine to citrulline and NO. F ~ . A..-- et al, BiochPmi~An~ phn~ v 1991, 42, 1849-1857. While t!ne role of NO as a signaling molecule in tlne ~;m~ n of guanylate cyclase is well I -1.1~AI.P~ r~ - ~ et al, 20 PhA----- ' 1 RPviews 1991, 43, 109-142), the origins of its cytoxicity remained unclear.
Recently a body of rnnnrPlline evidence surfaced which teaches that NO by itself may not be rPRr~nQi~' for cell damage (See Absts. of 1st Annual Mtg. of Oxygen Society, Nov. 124, 1993, 25 ChArlPe~n, SC, "Nitric Oxide Requires Suue~w~idt to E~ert RA~ ri-~-lAl Activity" by L. Brunnelli and J.S. Berl~mAn) Instead a more reactive species, p~lu~L~ iL iLe~ produced by the reattio~ of Du,u~.u. ide and NO, i8 found to play a role in the Cy-tOtOXiCity observed with the over-~-udu~Liu-- of NO. r~. U~ylL~ is known to 33 via a process which is first order in protons. The rate of proton catalyzed ~lf-~f l o~ n of p~.u..yl iL-ile (h~ . "the natural bL.L~;- uuu.d rate of decay") is u-~. DLuod from its study over a variety of pH rarlges (see;Keith et al. J ~hPm ~'~oc (A). p.90, 1969). When the pEI is 7.4 and the ~ i9 ~ d at 37 C, the observed rate for 35 the flflr ~ nc. l ~ of ~. u~y~ iLe is 3.6 x 10-1 sec-1 (see Beckman et al.
Proc. Natl. A~A-1 S~i U,'~ Vol 87, ppl620-1624, 1990). Beckman shows W095/31197 2 l 8 9 5 2 ~
.

that ~,.u~y~LilLiLe ~ generates a strong oAidant v.~ith reactivity similar to hydroAyl radical, as assessed by the oAidation of deo~ribose or dimethyl sulfoxide with the further s~c~ An that bu~i.uAid~ ~' ' protects vascular tissue si~m~ tpd to produce 5 s~ ,.uAidc and NO- under r ~ by ~ ,.lLLLg the fArm~t~An ûf p~.u,.y.LiLLiLe. &e Beckman et al, "Apparent HydroAyl Radical Pl~udu.,Lv~ by r~. UAylLLL iL~. T...I.l;. A ~..1_ for T~n~lAthpl~
Injury from Nitric OAide and Su~.UAid~" in Proc. ~t.l Arn~ Sri USA, Vol. 87, pp 1629-1624, February 1990.
10 Further, it i8 well '-liRh~cl that ~.u~.y~ liL~ ,1" "'~ to give the hydroAyl radical and nitrogen dioAide, a potent nitrating agent. Both of the6e species are potent oAidants shown to react with lipid --~--.~-t---~ and 6ulfhydryl moieties (See Radi et al "r~.UAylLiLLi Oxidation of Sulfhydryls" in The Journ~l Af Biol-A.~ir~ h~mietrv. Vol.
15 266, No. 7 March 6, pp 4244~250,1991).
Hardy et al suggest the int~rr~ t;AA of 2 with nitric oAide forms p~.uAy.,iL.ii.e or the protonation of 2 to form p~Ly1~uAyl radical is involved in the neutrophil~ .l killing of HAE cells (FASEB Meeting on April 5-9, 1992 in Anaheim, l~lifi~rni~) and further Hardy et al suggest a role for p~.uAylliLlik: in oxidative damage of human ~ l cells (Abstract in the "F~l- . ;.. ~1 Biology"
section of FASEB on March 28-April 1, 1993 in New Orlean3, LA).
In other words, harmful products from ptluAylliL-iLe ~lr~v~ c~- l AA is grerifirsllly taught by many .~ 3.
25 In addition, it hAs been shown that the reaction of pe:~u~ylliLliLt~ with Mn and Fe SOD results in il~Li~.Lu.l of the enzyme (See also Radi et al, A~rh P ' Biûrh.vs.. l9gl, 288, 481~87). It is now knov~n that p~.uAylliLIiL~ will also il~Li~ate CuZn SOD.
Thus, the effects of the ~F ,-~ ;I:An of p~.u~.~-liLlilæ, 3'' whether by the generation of damaging ~ A- I --. products or inactivation of SOD, in a wide variety of diseases are well ~l~.~...". .,I..
For eAample, a study assessing the A~rll t- :----c effects of p~.uAyllilliL~ on the rat colon is reported by Rachmilewitz et al in ''r~,uAy,,iL.iLe-induced Rat Colitis: A New Model of Colonic 35 Tlln~ from (~ Lt~ Y 105 (6)1993.
_ _ _ _ _ _ W0 9v/31197 2 1 8 9 5 2 8 F~
Beckma~ et al in PCT/US9V07894 (.,v.~ " l;~ e to U.S.
Patent No. 5,277,908) teac'n, specificaily that ~ vAy~iiL iLG is formed by the reaction of Du~ u~idv (2 ) and nitric oxide in tissues subjected to ische~iic, ;..n~ ûr septic ~ - -s Beci~an et al link SOD
.~v ~ :r~ rn and F,v.u ~, iL iLe to v~Ju~ut~u~;c lateral sclerosis (ALS) in ~, Vol 364, 12 August 1993 and Hogg et al and Beckman et al., cvli~Gly~ present a ,~ , between ~u~yuiliiL-G anc'i ~11. v,, 1. . usis in Birrh~ zil Sûciety l~nc~rt;nnc. Vol. 21, received December 22, 1992 and in "Extensive Nitration of Protein Tyrosines in 10 ~Iuman Atherosclerosis Detected by T- - - ~ rl ~
ChPm ~ e-Seyler. Vol. 375, pp 81-88, February 1994. Further, the hl~vl~ i of ~v~u~ P~LLilG in various disease states is found for lung diseases ~ d to cigarette smoke, ~ ,UvCl~.UDiS, vluyuL~u~hlc lateral sclerosis, cold-induced brain edema in (lhPm R.PC Trlrirrl lv Vol. 5, No. 3, 1992 pp 425-431. See also "Cold-induced Braii Ede_a in Mice" in The J-mrn~l of Bi-llr.~ir~l ChPmiQt~y. Vol.268, No. 21 Issue of July 25, pp 1539~15398, 1993.
More recently a spinal neuron toxicity assay has been developed by Scherch et al to screen for drugs which block p~lrvJ~.yllil.~i~G
2v tosicity. (23rd Annual Meeting of the Society for ~Lv~
Washington, D.D., Nove~nber 7-12, 1993 and ,h- ~ d in SrriPtv for N~ n~ Ahot-~rtq 19 (1-3) 1993 and ~ 94:4951.
Further, by ~ LiUg inactivation of SOD by reducing the presence of p~.u.~y~ iLLiLG the present invention also provides 25 Pnh~nr~pmpnt of known pl.~Oiolûgical benefits of DU,UGiU~de 1;.
in the L-, ' ' of diseases based on such benefits. In this regard SOD
and its mimics have been shown tû be useful in the L. GV- LLUG..I of diseases for the inhibition of an U~G~u~uducLv~ of OUI~GlV idv and nitric oxide. Thus, the present invention relates to the known L-~ for 3nv diseases by SOD and SOD _imics.
The Beckmvn et vl PCT ,~ alsû teaches that SODs catalyze the 1 ~ of the osygen radical Ou~ u~v and provides .G..cev which show SOD and variants thereof have been co~nonly utilized to prevent ûr reduce ûxidation injury in the IlGv~Ll~Glll of stroke 35 and head trauma, Luyu~,v...lial i6chemia, ~hd~min~l vascular wo 95131197 2 1 8 9 5 2 8 r~.,u~ ~r oeclusion, ey6titis, and a variety of ;.. n .. ".. ~ ,.. ~;1 .. c Beekman et al PCT Ar~lirAtirln also IG~Ui,lli~Go the presenee of p~.vAy lii, ile in these same di6ease '---~ -.C Al-r~. Al~d with 2- without ,...l;r~
the further illllJ-U..,~Gll~D of the present invention.
5 Further teaehings to the diseases known to be r~ - ~ d with 1,- ~,..I,Ill.,.li, by SOD or its ~imies are found in EP PuLli~Liu~ No. 0524161 (EP Appl. No. 92870097) whieh is i~ JUI ~ by referenee therefor.
Pûrphyrin , l . are diselosed in U.S. Patent No.
5,284,674 as valuable ~ r,,~, .r- r :r and Ll ~ agents, non-peptide 10 r,h.~r .l.kl ~:de analogs are diselosed in Japanese Patent pllhlirPt;rn Hei 5-331063 as Pn-lrJcPrinp reeeptor r-'tgr~ ul~llu~Jul~ ylhlD
are diselosed in U.S. Patent 5,286,474 to be valuable for locating and vicllAli7;ne mnmmAliAn tumor tissue and similar nitrogen l '~ ; e l"F"Iu~ ~IeD V~ithout a rmnpl^ Pd metl are diselosed as CytotoAie 15 agents in U.S. Patent No. 5 ~R3 rJ~ No metl rnmple~Pc and their ~cr-fillnPcc are ghown as now found in the present invention.
Metal; , ' - are, however, shown to be useful - - l-u~
in Derwent Abstract as ;- l~. .- F l;-t~s in JP05277377-A and MRI agents in U.S. Patent No. 5,284,944; eyan pigments in U.S. Patent No.
2û 5 ~R6,59~ r~ oc . l l i ~ ., phthalocyanine . .~ in U.S. Patent No. 5,283,146; a recording layer in an optical reeording medium in U.S.
Patent No. 5,284,943 and near infrared absorbers and diD~lay/l~ ùl.lhlg materials in an abstraet for U.S. 5 '~q6 1 a~
Iron ~ ;ll is diselosed to be an effeetive agent to bind 25 or oxidize nitric oxide which has a ~ .y ' ~, ' effeet when induced by a cytokine or by endotoxin for the l~ e aL~ , of diseases sueh as 6eptic shock in PCT ~ : -, No. PCT/US93/01288 (PuLli~i;iu.. No. WO 93/16721).
Other , 1 and their utilities are disclosed. For 30 example, ~R--Il-- --;----- Phthalocyanines" are disclosed as water soluble agents for ph~ yll~ic cancer Therapy in PlAtinllm ~ptAlrl Rev.
1995, 39, (1), 14-18; selected metllo-organic ~ , l are diselosed as ; of ;.. n .. ~i: -. in U.S. Patent No. 4, 866,054; Porphyrin and phthalocyanine antiviral (0..,l.n~ ..,c are di6closed as inhihitn~ Of 35 infection or replication of HIV in U.S. Patent No. 5,109,016; MAneAnPce wo 9~/31197 meso-tetra(1 ~ I)porphine are ~,yllLIIc~ and used as Lu~uo. -uA~ Li~ MRI contrast sgents; an abstract for JP 03273082 teaches peroxide-~oO J- ~ metal ~u~,u~ly~ S for uge as ~
in the . . .A ~ r~- I . . ~ of foods or other products; U.S. Patent No. 4758429 5 teaches iron L~ l porphyrin sulfonate acetate for ~Li~aLillg magnetic or electrical dipoles in the joint with an Al~ e el~LI ~ c field to treat arthritis and non-infectious joint diseases; an abstract of EP 392666 shows a non-toAic labile metal atom or comples such as 1,6,9,13-LtL~ - for use in the 10 L.~.,LII c.IL of a virus such as HlV. CA 119:203240 discloses selected mPtAll.~ ,u~l~lills as I~UU~ C~I~iC~ are found in French Patent NO.
91-6174. NU...~.UUb prlrlit;rlnAl l~.f; .ell~ 3 indicate ~nAlrgo mA1 U8es for metal crnnrlPYPC
Finally, nitrogen .~ g selected I~ IU~J~ are shown 15 in JP05331063 as ~ 1;" receptor A~ for treating and preventing ll~.i.iL.I~;o~l~ acute renal failure, I A~d;G~ u~c.Llly and .yu~ldiàl infarction.
,~llmmAry of thP Invent;rn The present invention is a method of treating a disease which ;0 iS ~ L~ 1Y affected by ~ A~ . of p~,.u~yll,LIiLe which is A. . . 1. . ,~ I Pt1 over, ie above or more than, a natural b~Lol uulld rate of decay in humans suffering from the disease ~U u~UI;~ lg ~
a crnnrolln~ or rrmro m~ which is a metal comple~ whereby the p~.u~lliLI;L~ is ~ u~e~ PILf~U~IY p.,.UA,~ iS A~ d to 25 a benign species. The ~ -u ~ is a liOand structure providing a cA~np'^~Pd metal, such as one of the transition r~etals, such as Mn, Fe, M and V. Preferred ligands are ll~&~u~ .l;c ligands, such as pUl,Ull~l;llS, aza ~ua~,~u~l~s and the like.
The present invention is a novel methûd of treating a disease 30 in ml~mm1l1c, including humans, ad~ LhO~Iu~ly affected by the absence of pi lu~ylliLIiL~ rnmrricinE A~ of an ~c~
A~ rr~ ... effective amount of a crmro~m~l of the formula

2 1 8 9 ~ 2 8 - -SL, I
Rt2~R3 Z
R11~5 R~ R~
~D R7 wherein R3, R6, Rg or Rl2 are; A IJ A_ Ily selected a group ~ of H, alkyl, alkenyl, CH2COOH, phenyl, pyridinyl, and N-alhyl~ lyl such that phenyl, pyridinyl 15 and N-alkyl~ l are ~ Phenyl ¢~9 Pyridyl N AL~yll~J
R
which are attached at a carbon atom, and 25 wherein phenyl is optionally .~ 'd by halogen, alkyl, aryl, ben2yl, COOH, CONH2, SO3H, NO2, NH2, N(R)3+, wherein R is hydrogen, alkyl, or alkylaryl;
pyridinyl is optionally s~ l Pd by halogen, alkyl, aryl, ben2yl, COOH CONH2, SOgH, NO2, NH2, N(Rh+
or NHCOR' wherein R is as defined above and R' is alkyl; and N-all~ .;L..~i ring is optionally ~ :l ,t~ d by halogen, alkyl, aryl, ben2yl, COOH, CONH2, SO3H, NO2, NH2, N(R)3+ or NHCOR' wherein R
35 and R' are as defined above;

woss/3lls7 21 8q528 Rl, R2, ~, R6, R7, R8, Rlo, or Rll are i--A~ -lly selected a group ~U~L,Lu.g of H, alkyl, alkenyl, carbû~yalkyl, ~1, Br, F, N02, hydroxyalkyl, and SOgH or Rl and R2 ca~l be taken together to form a ring of from 5 to 8 carbon6 ,u~f~ bly 6;
5 X and Y are suitable ligands or charge-n - ~ -l; . e anions which are derived from any ,--~ or pulyd~ Lill,e L,uuli'- " e ligand or ligand system or the Cull ~ A~ e anion thereof (for e~a nple benzoic acid or benzoate a~ion, phenol or r'~- .. ;Ar anion, alcohol or alko~ide anion) and are i~A~ IJ. .~A_~I.ly selectea from the group rnnQio~ine of ~D halide, oxo, aquo, hydroxo, alcohol, phenol, dio~ygen, peroxo, hy~Luuelu~u, alkylperoxo, a.~l,u~,.u,.u, ammonia, alkylamino, arylamino, heL~,.u~ oalkyl amino, heterocycloaryl, amino, amine oxides, hydrazine, alkyl L~lla~llc, aryl hydrazine, nitric o~ide, cyanide, cyanate, LluO~ a~laLe~ iL7u~ aL~Lê~ ;E_" ' .ralLàLe, alkyl nitrile, L5 aryl nitrile, alkyl isonitrile, aryl isonitrile, nitrate, nitrite, azido, alkyl sulfonic acid, aryl sulfonic acid, alkyl sulfo~ide, aryl sulfo~ide, alkyl aryl su'Lfoxide, a'kyl su'lfenic acid, aryl su'lfenic acid, a'lkyl su'lfinic acid, aryl su7L-fi~lic acid, a'~kyl thiol cal7uu.yLc acid, aryl t'niol ~al7uu.yLc acid, a'Lkyl thiol Ll ioc&,'uu.ylic acid, aryl thiol L~ iû~ uu~ylic acid, a'ikyl~ ~ai7uu~.ylic acid (8UC7~ as acetic acid, LLinu-,.~ - acid, oxa'Lic acid), - aryl ~al7uu~LyLc acid (suc'n as benzoic acid, phtha'Lic acid), urea, a'lkyl urea, aryl urea, a'lkyl aryl urea, thiourea, a'ikyl thiourea, aryl t'~iourea, alkyl aryl t'niourea, su'lfate, su'ifite, bisu'Lfate, bisulfite, thinR..lfs7tP, thirql.lfitP, hydrosu'fite, alkyl rl. ~ l ;, P aryl rl, ?l l; P, a'lkyl 25 rhncrhinP oxide, aryl rl~r$11 i, r oiide, a'lkyl aryl rl~r- l,;~,r oxide, a'kyl rhAsrhinP su'lfide, aryl rhnqFhinP su'Lfide, a'ikyl aryl rhncrhin~
su'Lfide, a'Lkyl ~I- ,L h :r acid, a~yl rl~ ;c acid, alkyl rl7~ol 1~; c acid, aryl rl ~ ~l k ~:r acid, a'Lkyl r~ L k; C acid, aryl 1 acid, rhnsrhAte, ll; ~l~h~ t~ rhncrhitP ~ h~
30 L., ho~ , hydrogen rh~ - lJ ~ , dihydrogen ~ A~ P a'ikyl ~,U ULidi..o, a~yl guanidino, a'Lkyl aryl guanidino, alkyl ~aLbaLIlaLe, aryl C~L~7UL7111aLe, a'ikyl aryl ca~lJ~uaLe~ a'Lkyl 1.1-;-,- .1. ..-1~, aryl L7LLiu~_~baLuaLe, a'lkyl aryl LlLiG~a~7uL7~-Le~ a'ikyl dithio.,_L'u~_l~, aryl diLLIiG~aLbaLuaLê~ alkyl aryl diLlLiU~aL7UaLi aLe, 7u;ual'uullaLe~ carbonate, 36 perch~orate, chlorate, cblorite, Lypo~hln~ite. ~.'u.u~aLe, bromate, wo 95/31197 2 1 8 9 ~ 2 8 r~ j" ~, bromite, l~uL.ulluLe, tetr~h~ L~luu~ùLul~e, h~,~lluû,u~ , hexafluu,~ , IIY,U~ iûdate, periodate, r- ' I ' 3, tetr~aryl bûrate, tetra alkyl borate, tartrate, salicylate, E ~rinofe, citrate, ascorbate, I--r l _~ IP, amino acid, 5 L~d,u~ur~ acid, Ll-,ûlu~yl~e, and anions of ion exchange resins, or systems; with the proviso that when the X and Y - I ~ ; - ~ complex has a net positive charge then Z is present and is a counter ion which is ;".l. lJ~ ly X or Y, or when the X and Y r...,~ complex has net negative charge then Z is present and is a counter ion selected from a 10 group ~ e of alkaline and alkaline earth cations, organic cations such as alkyl or alkylaryl --.. -.. ;.. cations; and M is selected from the group ~ of Mn, Fe, Ni and V;
St~, ' e II
Rz R3 Rl~ R4 2~)z ~
~; ~ ~
R1z ~ Rg wherein 30 R' is CH or N;
Rl, R2, R3, R4, Rs, R6, R7, Rg, Rg, Rlo, Rll, Rl2, Rl3, Rl4, Rls~ and Rlg are ;,~ -lly selected from a group consisting of H, SO3H, COOH, NO2, N~I2. and N-al~l~o, X, Y, Z and M are selected as defined above;

_ _ _ ~095J31197 21 89528 m R~ R,~j~R~
--~N~ ~ "N~R4 Z
R,~ Rl5 ~ R R5 Rtz;~N `N'~
R10, R~.
A

wherein Rl, Rs, Rg, and Rl3 are; ~ y a direct bond or CH2;
15 R2, R2', R4, R4', R6, R6', Rg, Rg', Rlo, Rlo', Rl2~ R12~ Rl4, R14, Rl6, Rl6' are in~PrPnrlPnt.ly EI, or alkyl;
Ra, R7, R1l, R1s are ;..~ IJ .~ I.y H or alkyl;
X, Y, Z and M are as defined above;
2D R14~R1 j~R2~
R~ 3~N ~ N~R4, Z R1~ ~YJ~R6 Rg B

wherein R1, Rs, R3, and R12 are ;"~IJ~ lly a direct bond or CH2;
R2. ~2'.R4. R4',R6, R6', R7, R3, Rg', Rll, Rll', RlS, Rl3', Rl4 are . ..I.ly H or alkyl;
R3 and R1o are in~ tly H or alkyl;
X, Y, Z and M are as defined above;

R1 41~ 1 ~R2' R13~N ~ N=~R3 R12 `,M~ R4 Z R~ R
Rg wherein C
~D Rl, R4, Rg, Rl2 are in~ .A~ ~ly a direct bond or CH2;
R2, R2', R3, Rs, Rs', R7, R3, R3', Rll, Rll', Rl3, Rl3', Rl4 are ;"A~ ."1~ ,lly H or alkyl;
Rlo is H or alkyl;
X, Y, Z and M are as defined above;
L~ Rtz Rl~Rz~
R~ ~
R8~ RlR6 wherein 25 Rl, R~, R7 and Rlo are ;...1. l~. ..~_..1.1~ a direct bond or CH2;
R2, R2', R3, Rs, Rs', R6, Rg, Rg', R3, Rll, Rll' and Rl2 are in~lPrPnAPntly H or alkyl;
X, Y, Z and M are as defined above;

WO9!i/31197 21 89528 wherein 10 Rl, R4, Rs and Rll are intl~ y a direct bond or CH2;
R2, R3, R3', Rs, Rs', R7, R7', ~2~9, Rlo, Rlo', Rl2, R12' arld Rl3 are ;".1~1._..-1. ..I.ly H or alkyl;
R6 i~ hydrogerl and alkyl;
X, Y, Z and M are as defined above;

?~ 6~
2D R9'~N y3~R5 R~ R7 6 wherein 25 Rl, R4, R7 and Rlo are int~ .. lly H or alkyl;
R2, R3, R3', R5, R5', R5, R8, lE2~, R3', Rll, Rl,' and Rl2 are ;"tl_l._..tl_.~.ly H or alkyl;
X, Y, Z and M are as defined above;
9~ :

W0 95~3119~ 2 1 8 9 5 2 8 P~ S

ZRs ~ ~ R2 R~/~R3 G

10 wherein Rl, R3, R4 and R6 are inrl- IJ~ Ily H or alkyl;
R2 and Rs are ;~ ly selected from the group consisting of H, alkyl, SO3H, NO2, NH2, halogen, COOH, and N(R)3+
wherein R is as defined above;
~5 X, Y, Z and M are as defined above;
:,_'< Z
wherein Rl, R2, R3, R4 are ;..rl-u~..rl~..lly selected from the group 3D CUIls;DlAi~.g of H, alkyl, SO3H, NO2, NH2, halogen, COOH and N(R)3+
wherein R is as defined aboYe;
X, Y, Z and M are as defined above;
.
~5 W0 9513 1 197 I ~ I ~ IJ .. ' .

~L ~
R7 R7.
R6' X I R6~
R4~t ~ N=<~ ~R4-R3~ 0-- o~R3-wherein Rl, Rl', R2, R2', R3, R3', R4, R4', R5, R5', R6, R6', R7 and R7' l~v~ are ;., ~ A ~lly selected from a ~roup ~ gt of H, alkyl, alkoxy, NO2, aryl, halogen, NH2, S03H, and R6, R6', R7 and R7' may each be taken together with one other of R6, R6', R7 and R7' to form a cyclic group, lul~f~., bly a 6 carbon cycloalkyl group;
Ml is Fe, Ni or V;
15 X, Y and Z are as defined above together vrith a l~l~l . . ._. F,. . l :. ~lly e.( , l l carrier, ~u~ y in unit dosage form.
The present invention i8 also a r~ r~
for the l,ltàl~ of a di6esse in humans ~d~ u.~ly affected by ,-l~ .. " I,~d AF ~ l: . over the natural bL 4;l uul~d rate of decay of ~e~lU~ylliLliL~ Cul.l~l;D;IIg an amount effective for the ~
~l~r.. l.t~ of p.. ù~yluL~ in humans of a r ~l-u .. ,A of the formula I, II, IIIA, IIIB, IIIC, IIID, IIIE, mF, IIIG, IIIH as defined above with a pl.~ r. -I t_lly pt- ~ e carrier in unit dosage form, ~ul~f~,.a~ly oral unit dosage form.
25 X, Y and Z are each a IJ-~, F~ lly ~t~Dpt-~l- anion or cation.
RriDf D~ . . ;u~ivll of t.hP Drawin~
FIGIJR.F. 1: Plot of kob~ vs cat~lysts ~ullcci.lLa~ivll for Fe(III)TMPS and 3v Fe(III)TPPS il~ tr ~;nE catalytic nature of ~FC~ I of pc~u~ylli~ by metal, , 1 FIGUR.li. 2- Plot il1llrtrs~tinE the inactlvation of CuZn~OD by p~,.vl.y.li~ e.

W095131197 ~ 8 FMURTt' 3: Plot ill~ the ~ " ---lA-.~ vL~_Lu., of CuZnSOD against il~ ,LvaLo~ by p~.u...~, liL iLe using p~UA~ . iLI-iLd .V~ An catalysts Fe(III)TMPyP.
5 FiGVRTt'. 4: Plot ~ e the ~ : , A_l,_"~ protection of CuZnSOD against i~-a~L~aL~.. by ~,u~ylliL~ile using p_,u~y liL-iL~
A~r~.. .l..~-:l:An catalyst Fe(III)TMPS.
FIGlJRTt'. 5: r.i,u..~, iL.iLe-mediated human , i~u.t s~ulsr Pn~lntllPli~l lv cell injury. AllthPnti~ p~,u~ illiL~ was overlaid directly ûnto to 5lCr-labeled HMDE cells grown in 96-well cell cv1ture plates. After 45 min, the amount of specific cell injury was A~ V and cu..~l~t~3~ tA
p~U--Y- iL-iL~3 .,u..c~.L-aLion by least squares .t~ ' line. Values represent the average of three replicates +/- SEM.
FIGURli', 6: rt:~u.~ylliLIiLt catalysts, Fe(TMPyP) (triangle) and ~Ti(TT)~i~nA~4)PF6( circle ) were added tA HDME cells in the cell injury assay imm~ tPly before the addition of authentic p~,lU~.y. iL.iLe. After 45 min, the amount of specific cell injury was assessed by the amount 2u of radiolabel released into the medium. Values represent the average of three replicas +/- SEM. ~p<0.01 vs. 0 uM control by Dunnett's t Test.
FIGURF, 7: Tnhihition of neutrophil-mediated injury to human aortic 1;A1 cells by Fe(TMPyP). r~,,u~. iL-iLe catalyst, Fe(TMPyP), 25 was added to neutrophils in the cell irljury assay irAnAn~ tPly before activation by TNF/C5a. After 2 h, the amount of specific cell injury was assessed by the amount of, ~ 1 1 3l released into the medium. Values represent the average of three replicas +/- SEM. ~p~0.01 vs. 0 uM
control by Dunnett's t Test.

FIGTJ~Tt', 8: C~ of Ni and Fe Catalyst Protection ûf RAW Cells from PN(p~.u~.y. iL iLu)-mediated Injury. RAW 264.7 cells were plated at a~ u. hllaL~ly 2x105 per well of a 96-well plate. PN(360 ~ u lùl&l) was added to every well of cells in the presence of i...,. ~ ..g 35 cu..~i..L. ~lLu.ls of Ni catalyst or FeTMPyP resulting in total protection _ _ woss/3lls7 2~ 8q528 16 from PN-mediated injury as ~ d by t.'le ability of cells to Alamsr Blue to a n~u....~ll. product. Each condition ..L~ t,he mean of 4 wells +se~n.
5 FIGUR~ 9: Protection from PN-mediated RAW Cell Injury by Fe Catalysts. Cells were treated with 500 micromolsr PN in ti'~e presence or the absence of tl'le following catalysts: Fe~MPyP, FeTMPS, FeTPPS.
Cell viability was I. v,..lo.~vl as described in the text and figure legends 1, 2 and 3. Vslues represent tl'ie mean of 4 ~ r ---;-~ fi +sem.
FIGUR~: 10 . Effects of FeTMPS, FeTMPyP ûr ZnTMPyP (30 mg/kg, i.v bolus) ~ ed 3 h after challenge with E. coli l;l,U~U~ V~. l,... :A_ (LPS, 3 mg/kg, i.v bolus) on the increase in lea. age of " '-' -'l~d slbumin (plasma e,.L-~ ]/g tissue) observed 1 h later (e.g 4 h 15 after LPS challenge) in the rat jejunurn. Results are shown as mesnis.e.m of ~8 rats.
Det ilAd D~.,c~.liul, nf th~ Inv~ntinn 2~ As utilized herein, the term ~alkyl", alone or in . ~
means a straight-cihain or braiched-chain slkyl radical ~ I.A;";..e from 1 to about 22 carborl atoms, u..if..~.blr from about 1 to about 18 carbon atoms, and most preferably from about 1 to about 12 carbon atoms. Examples of such radicals include, but are not limited to 25 methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, iso-amyl, he~yl, octyl, nonyl, decyl, dodecyl, tetradecyl, he~adecyl, octadecyl and eicosyl. The term aryl, alone or in c~ hinAt;~ mean8 a phenyl or naphti'lyl radical which optionally carries one or more ~ selected from alkyl, cycloalkyl, 3n ~ nyl, aryl, h_~.u. ~lc, slkoxyaryl, alkaryl, alkoxy, i'alogen, hydroxy, amine, cyano, nitro, alkylthio, phenoxy, ether, L.illuu.u.~ l and t'e like, such as phenyl, p-tolyl, 4-methoxy-phenyl, ~(tert-butoxy)phenyl, 4-lluu~u,uh~ l, 1 cLlv~vluh~ l, 4-l~ .u~,JI._.l~l, 1-naphthyl, 2-naphthyl, and the like. The term "aralkyl, alone or in 35 ~nmhin~tinn, meang an alkyl or cycloalkyl radical as defined herein in which one hydrogen atom is replaced by an aryl radical as defined W0 95/31197 2 1 8 9 5 2 8 E~

herein, such as benzyl, 2-phenylethyl, and the like. The term h~L~.u~"clic~ means ring sLlu~,luu~ at least one other kind of atom, in addition to carbon, in the ring The most common of the other kinds of atoms include nitrogen, oxygen and sulrur. F l of 5 htl~.u.,.~lics include, but are not limited to, ~.~..oLdu..~l, piperidyl, in~ flif~...yl, tetral~.l-vruuyl, vk~ L~ '' yl, furyl, thienyl, pyridyl, quinûlyl, i~uf.~uillfJIyl~ ,u.~;d~iuyl~ pyrazinyl, indolyl, hl id~ulyl, o~azolyl, thiazolyl, pyrazolyl, pyridinyl, ~ yl, l~l, triazolyl and tetrazolyl groups. The term IL cycloalkyln, alone or in c-^nnhin~t;^n means a cycloalkyl radical rrom 3 to about 10, ~ ly from 3 to about 8, and most U1G~ IY from 3 to about 6 carbon atoms Examples of such cycloalkyl radicals include, but are not limited to, .,.~lulJ~u~yl, ~,~lo~uLyl, ~,luyll_vyl, ~ lùll~yl, L~lùll~:~Lyl, cyclooctyl, and p~,.L.~dlu~,u~Lh.~l.
35 The term cycloalkenyln, alone or in r^mhin~tin^ means a cycloalkyl radical hsYing one or more double bonds. Esamples of cyrln~ll yl radicals include, but are not limited to ~.l.~ ' .,1, ~IûL~ ..~...~l, .,lûoo~L~"yl, ~ iPnyl, cy~^1n~ f3ipnyl~ and cyrlf)o~t^~iPnyl.
The ~a~.lu~,~,Lc ligands useful in the present inYention 2D wherein the formula is Structure I can be prepared according to the generv. synthetic methods known in the art for ".~ ..Lu-- of certain ligands. See, for example, 1) CJ"..,U^ 1 -." S.; Meunier, B. Inorg. Chem. 31, 1999^2006, (1992).
2)Robert, A.; Loock, B.; '~ M.; Meunier, B. Inorg Chem. 30, 25 706-711, (1991).

3)Lindsey, J. S.; Wagner, R.W.J. Org.Chem. 54, 828-836, (1989).

4) Zipplies, M.F.; Lee, W.A.;Bruice, T.C. J. Am. Chem. Soc. 108, 4433 4445, (1986).
The Luc~lu~ Lc ligands useful in the present invention 30 wherein the formula is Structure II can be prepared according to the general synthetic methods known in the art for ~ ~ph~Lu~ ûf certain ligands. See, for example, 1) Some r- .~l~v ~ are ~,ù~ ..,;ally aYailable from ruL~l~yli Products, Inc. (Logan, Utah.) 35 2) Y. L. Meltze; Phthalocyanine T ' ~'~y in Chemical Process ReYiews No. 42.; Noyes Data Corp, Park Ridge, N.J. (1970).

WO95/31197 2 1 8q 528 The Lu~.u~ic ligands useful in the present invention wherein the formula i8 Structure III can be prepared according tc~ the general synthetic methods known in the art for p~ JalaLiull of certain ligands. See, for es~ample, 6 l)Goedken, V. L.; Molin-Cass, J.; Whang, Y-A; J.C.S.Chem.Comm.
337-338, (1973) 2) Martin, J.G.; ~llmmin~, S.C.; Inorg.Chem. 12, 1477-1482, (1973).
3)Riley, D.P.; Stone, J~; Busch, D.H. J Am ~hP n ~oe. 98, 1752-1762, (1976).
10 4)Dabrowiak, J.C.; Merrell, P.H.; Stone, J A.; Busch, D.H.;
J.Am.t~h~m ~oc 95, 6613-6622, (197 3).

5) Riley, D.P.; Busch, D.H.; Inorg. Chem. 23, 3235-3241, (1984).

6)Watkins, D.D.; Riley, D.P.; Stone, J.A.; Busch, D.H.; Inorg. Chem.
15, 387-393, (1976).
15 7)Riley, D.P.; Stone, J.A.; Busch, D.H.; J.Am.ChPm ~oe 99, 767-777, (1977).
The i..ac,v~,J.lic ligands useful in the present invention wherein the formula is Structure lV can be prepared according to the general synthetic methods known in the art for ~ laliùll of certain Z' ligands. See, for example, 1) Diehl, H.; Hoch, C.C.; Inorganic Synthesis Vol 3. p 196.
McGraw-Hill, New York (1950).
2) Srinivasan, K; Michaud, P.; Kochi, J.K; J. A~ Ch~m ~oc. 108, 230~2320, (1986).
25 3) Samsel, E.G.; Srinivasan, K; Kochi, J.K J. Am.Chem. Soc. 107, 7606-7617, (1985).
I~e c- .l v . ~l~ of the present invention can possess one or more &b.yll~-~,LI ;C carbon atoms and are thus capable of escisting in the form ûf optical isomers as well as in the form of racemic or nu~ _v Lc 3n misctures thereof. The optical isûmers can be obtained by rPc~ n of the racemic misctures according to conventional processes, for example by formation of ~li&,DL~ - - salts by LlvaLLv~.lL with an optically active acid. Examples of ~ vlu~;aLv acids are tartaric, LacvL~lLmL~I;c, Llv~v~LnLLaLic~ diLvluu~lLmLnl;c and 35 ~Dlulvhu.Dulfonic acid and then 6v~LIaLiull of the mixture of diabLvl~;Dvll.~.D by crysf~ 7~ n followed by bberation of the optically W0 95/31197 2 1 8 9 5 2 8 F~

active bsses from these salts. A different process for 8PrArp~;~l^, of optical isomers inYolves the use of a chiral cL-~ A~ Y column optimally chosen to maAimize the l;_~JQI '- of the I ,A ,I - ,l D. Still another aYailable method involves synthesis of covalent 5 ~ ^ mnlP~ PA by reacting one or more ~e~u~d~- v amine group( 8) of the ~ u ~ u ~ of the invention viith an optically pure acid in an activated form or an ûptically pure i6~ ~1n~e. The sv..LLe~.._d ~1;A ' ~.J;DV~ ..D can be separated by ;u--v~-Liu-~l means such as ~LulunL~f~1Q~ y~ liQt;llAAt;on, cryetA~ At;An or ,Q-lhlimAAt;^-^., and then 10 I-~Luly~cid to deliver the ~ frl1y pure lig nd. The optically active ~ - l-ov~ l^ of the inventiûn can likev~ise be obtained by utilizing optically active starting materials, such as natural amino acids.
To screen metal rornrl^YP~ for ue1uAy1P~I~;ie ~r~ rl catalytic activity of the present invention, p~.uAy~ ile is prepared and 35 isolated as its sodium salt by the reaction of acidic hydrogen peroxide viith sodium rlitrite followed by rapid ~ ._,.~1,;"~ with NaOH as set out by Halfpenny and Robinson, in J. ChPm Snr 1952, 928-g38.
r~.v,-~. il, iLrQ has an ~ nll~e ~ - at 302 nm with an P~tinrt~^n rA~llh -.1 of 1670 M-lcm-l. Therefore, it is possible to O directly observe the rlF .1~U^~ I ~^n of ~.uAylliLIiLe by stop-flow Du~,LI u~ f analysi6 by ^ - ;~ the f~r~ u~ n of the _~G~u1b~,e at 302 nm. That i8~ such observation of the d~ -I- - 1: . of pe.uAy. iL iL~ at a rate ~Acc~ .nLed over thAe natural rl~^ - ..1 G~;1: . rate with the addition of the metal comple~ identifies a c---..1., ..rl of the 26 present invention.
In addition, it is now found that ~_.u~y1lil1iL~ i-.a~,Lv ' -CuZnSOD enzyme in a ~- . - .1 . A~ -..~ tlP~^,Dnrl_nt m,A~nner. Since it is known p_.uAy- iL-iLe also i..~livc,l~i~ MnSOD (See "r~UA~ iLI-iL~-Mediated Tyrosine Nitration Catalyzed by Su,u_.u. idc Dismutase" by 30 Ischiropoulos et al in ArrhivPA ~f R;h- 1.. .~;r 1~ pn~l r I Vol.
298, No. 2, No. _ u~e. 1, pp. 431-437, 1992), the present invention prûvides a ~ u --.~l which protects CuZnSOD from inactivation by :luA.~iL iLe.
In this manner the r~mrUllnA of the present invention i6 35 shown to be useful in treating a disease in a human adv~ L~u~rly affected by the presence of the SOD enzyme.

WO 9S/31197 ~ 5 2 ~ IL~ _. ~
2n That i6, the L. c~l~c.~L of the present invention iB for a disease state either caused by the presence of a p~.u.~ iLe of caused by the lack of the protective presence of the SOD enz~nne such as in a ~u~.L~d infarct, stroke or an A~ ... -- .. r disease. These latter 5 diseases are also shown to be ae~ with the presence of p_.u~y~ iLIiLe.
These metal cnmpl^-oA are fûund to be within the prcsent invention by ~ -. of their ~ . effect on p.,.u..y, i~liLe as set out herein.
l~v~ C~ 1 equivalentA of the general formulas set forth above for the ~ -v ~ki and dl ~;va~ as well as the ;.-~ ....P.~
are ~ v~ A othervvi8e Cull- Al~ e thereto and having the same general p.u~v~.Lic~ such as LauLv~ D of the ~ -u~ c and such as wherein ûne or mûre of the various R groups are simple ~.ia~iull& of lv the fillhot;tllPnto as defined therein, e.g., wherein ~ D~ which are a higher alkyl group than that indicated, or where the tosyl groups are other nitrogon or oygen protecting groups or wherein the O-tosyl is a halide. Anions having a charge other than 1, e.g., carbonate, rhnRrh~t~, and hydrogen l~h.~ , can be used instead of anions Z' having a charge of 1, 80 long as they do not adversely affect the overall activity of the complex. However, u6ing anions having a charge other than 1 will result in a slight mn~ifir~ n of the general formula for the complex 6et forth above. In addition, where a ~lbn~;blPnt is as, or can be, a hydrogen, the exact chemical nab~re of a ~ 1-- ~:1 .. ., 25 which is other than hydrogen at that position, e.g., a llyvlv~lv,l radical or a halogen, hydroxy, amino and the like î....~ 1 group, is not cri~ical so long as it does not adversely affect the overall activity and/or synthesis procedure.
The chemical reactions shown by the ef~,L..c~ described above are generally disclosed in terms of va.ia~ivL,s appropriate for their broadest ,~ " to the ~ al~.~iu.. of the '1~0'.~.Ao of this invention. O, -lly, the reactions may not be ~r~lir~hl? as described to each cnmrol~n~i included within the disclosed scope. The rnnnpo~ln/ic for which this occurs will be readily IL ù~;. i..~d by those 35 skilled in the art. In all such cases, either the reactions can be g~ cPccfillly p_ f.. ~ by ~u~ Liu~lal mnrlifir~tinnno known to those

7 ~ 1 8~ r~ J~ -skilled in the art, e.g., by Alu,ul u~ .;aLe protection of ~ - f~ e groups, by changing t4 alternative ~u..~-~.Liu..&l reagents, by routine ~- A~t?ifir?~t;AA of reaction ~----..l-l -~.,a, and the like, 4r other reactionsdisclosed herein or otherv~ise c~l.ve..Liu.lal, will be nrrli~ t4 the 5 ,u.~pL-~liu-- of the ~u,-c, ' C ~c ~ rl~ of this invention. In all ,u~ Li~., methods, all starting materials are known or rêadily preparable from known starting motPriola Without further Pl~h~ it is believed that one skilled in the art can, using the preceding rlParrirh~n~ utilize the present 10 invention t4 its fullest extent. The fûllûwing preferred 6pecific ,rJIl",~ are, therefore, t4 be cûnstrued as merely illustrative, and not lirnitative of the Ic U-i-ldcl of the disclosure in any way whatsoever.
~:lro~.lPa All reagents were used as received unless otherwise inrti~AtP~' 5.10,1fi,~0-tetrakis(N-Methyl-4-pyridyl)pû.~l.yiil- tetrat4sylate and Acetat4-5 10,1fi,~0-tetrakis(4-s~ll r... A~ yl),uùl,u~lylill iron(III) were 2û pul~b_d from Porphyrin Products Inc. (Logan, UT). Iron(III)citrate and iron(III)EDTA rornpl^-PR were ,uul~ B~d from Aldrich Chemical Cû. (Milwaukee, VVI). All nuclear magnetic ICI~UULI1~CC (NMR) spectra were obtained on Varian VXR-300 ûr Varian VXR-400 D,UO~,LlU~.D.
Qualitative and ~lu_.LiL~Li~c mass spectra were run on a Finnigan 25 MAT.^.0, a Finnigan 4600 and a VG40-250T D,u_~LIulu.~t~.D.
F.~mvlP 1 Sy. of ~ t (5 . 10 . 1 fi 0 tetrA k c(N m P ItYI 4 ,,vridvl )~u, ~ul iron (ITT) tetra-tncvl..tP Fe(HT)TMF'vP.
~1 0 1 fi."0-Tetra-(N-methyl-pyridyl)pori,hine .~Ll ~Lu~
3[ (H2TMF~yP) (0.30 g, 0.231 mmole) was charged t4 a 100 mL round bottom flask equipped with a magnetic stir bar and was dissolved in a minimal amount of MeOH. Anhydrous Fe(OAc)2 (0.120g, 0.692 mmole) was added followed ;....nP 1;AI~1Y by 25 rnL of glacial acetic acid nd 100 uL of Ll;-;LylA~ ue. The reaction mixture was heated to 35 reflu~. The reactiûn was l~o~liLu~d by visible D,Ue~l' ùSCu,uy and was wo g~/31197 2 1 8 9 5 2 8 r~

rd to be complete with the ~ u~ c of a strong band at 426 nm indicative of the m^lnllAtpd porphyrin. The MeOH was removed by O~a~uùl&Li~ A and the solid was taken up again in a minimal amount of MeOH. The miAsture was ~ d under vacuum to a total volume 5 of -20 mL at which point the lAnreacted Fe(OAc)2 ~ The - solid was separated by . ~ ;r ~G~ and the mother liquor i8 i,LIu,ua~u~ l.ed on a Sephade~ LH-20 columAn (2 x 30 cm) us~ng MeOH as eluent. The ir~itial colored band was collected aiAd Fe(m)l~yP(OAc) was isolated by ~ . after ,.ayuldLull of 10 solvent and L ;~,~u Lul. v~ith ether to jJive 85 mg ( 26%) of the desired product as ~^~nnfirrnPd by mass spectral analysis.
mr~^ 2 ~ 3vl^th~cicof5.10.1'i~n-tetr,ki~(3.5-~ r....r~l~.l..,...l.vi)Dulv n.^-t^cn~lillm s- lt (~2TMPS).
15 5,10,15,20-~LaAu~ yl~v~llyl ~ (H2TMP) was preparedby the .. ~l~."~l;^n of pyrrole and mPci~ lPhyde in sealed glass tubes by the method of Badger ( G.M. Badger, R.A. Jones, R.L. Laslett Auôt. J.
Chem., 17, 1022, tl964]) or in reflu~ing collidine according to the literature ~U-C~U~ ALvll of Meunier ( Meunier et. al. Nouv. J. Chim., 10, 2'' 39-49, [1986]). Chlorin; . . .I.., .; I: Ar were re~noved by o~idation with 2,3-dichloro-5,6-dicyano-1,4,-k .~70~ P in refiuxing benzene followed by ~LIu~a~Lalully on basic alumina. Both methods produced nearly identical yields of H2TMPS.
Synthesis of H2TMPS was achieved using a slight 25 mn~ifil~fi~ n of the method of Meunier (Meunier et. al. Inorg Chem, 31, 1999-2006, [1992]). A 25 mL rou~d botto3n 31ask equipped with a ref~u~c cuudt u~,~, and stir bar was charged with H2TMP (L0 g, 1.28 mmole). Oleum ( H2SO4 f 18-23% SO3) 10 mL wa3 added and the reaction was heated to 30 80 C for 40 3nin. The reaction was cooled and its contents wâs added dropwise to 100 rnL of water cooled in an ice bath. The resulting water solution wa3 neutralized with 2N NaOH (~220 mL) to a pH=6 7. The water was removed by ~a~uûlàLull and the resulting solid residue was triturated with a 3ninimal amûunt of MeOH. The resulting precipitate 35 was removed by f~ltration and the filtrate was furtLer ~ to W095131197 2 1 8 9 5 2 8 r~

60 mL under vacuum. The resulting p~ ;iaL~ ( ~A~A~it;~n~l Na2SO4) was separated by ~ The b ~ was ~.P~u,aLed to dryness ~ 1.59 g (78%) of the desired ~ .lr...~ pu~ ylhl.
E~amDle 3 5 SyrthPRic of ~rPtotn 5.10.15 ~Otetr~kiqt~ ;R~ ,V¦)UUIV1 Mf~ TTT) n~ m s~lt (Mn(ITT)TMPS).
H2TMPS ( 0.2 g, 0.125 mmole) and Mn(OAc)2 (0.296 g, 1.71 mmole) was dissolved in 38 mL of water and was heated to 85 C for 1 h.
The reaction was ~u. iLu.~,d by visible b"~_L.~ and was 10 flotPrrninPd to be complete when the Soret band ( 416 nm) of the free base porphyrin was replaced by a new band at 468 nm cl ala~L~IibLc of Mn(III) pO~,u~lylill specie6. The reaction was reduced in volume under vacuum to 10 mL and was ~LIU~aLU~ l~,ullc~ on a Dûwex 50~ 8 cation exchange resin (H+ form) to remove exce66 Mn(OAc)2. The eluent wa6 15 reduced in volume to 10 mL and was adju6ted to pH=8.0 with 1.0 N
NaOH. The re6ulting 601ution wa6 ~.ap~lat~ to dry-ne6s. The residue was taken up in 7 mL of MeOH and d.. ..Al,jC., 1~ on a Sephadex LH-20 column using MeOH as eluent. The purple band was collected and ~.a,uulahd to dry-ness giving 0.175 g(90%) of the desired mptoll~tpdû por~Ahyrin as A~ d by mass spectral analysis.
FYs~r,~Al~ 4 Syrlt.hPRiC Af~rPts~tn-5.lo.lfi ~n-tetr~kic(3.5-dicull'l...~l-....P~.lvl)uul~ v,l-- Irnn (TTT) G. ~ ... s~lt (Fe(lTnT~PS).
26 H2TMPS ( 0.2 g, 0.126 ~nole) and Fe(OAc)2 ( 0.300 gl.72 mmole) was dissolved in 38 r~L ûf water. The reaction mixture was brought to ref~ux and was ~u. iLul~ by visible s,u~,~LIuD~u,u~ tû
A~PtPrminP complete mPtqll~tlnn Upon rAmrl ' the reaction was filtered and reduced in volume to 10 mL. The orange-brown reaction - 3, mixture was passed through a Dowex 50WX-8 cation exchange column (H+ from) to remove excess Fe(OAc)2. The eluPnt was reduced in volume to 10 mL and was adjusted tA a pH= 7.5 with 1.0 N NaOH. The resulting 601ution was e.apulaL~d to dryness. The residue was taken up in 7 mL of MeOH and ~1.1 u~Logl a,ulled on a Sephadex LH-20 36 colu_n using MeOH as eluent. The orange-brown band wa6 W095/31197 2 1 8 9 5 2 8 ~

O~ied to dryness giving 0.170 g ( 72% ) of the desired Fe porphyin as confirmed by mass spectral analysis.
F,~ 5 SvnthPRiC nfArPtAtn fi ln.l~ n tetrAkicr3.s 5 dic-llf~...AI "",~ UL V1~ ., Nirkpl ~ri) ... I z. ~/..ll,.... gAlt (Ni(TT)TMPS).
H2TMPS ( 0.1 g, 0.063 mmole) and Ni(OAc)2 ( 0.156 g, 0.63 mmole) was dwgsûlved in 20 mL of water and was re~uxed for 3 h. The reaction ixture was ûrange in cûlor indicative of the Ni porphyrin.
10 The ~ , ~ ' of the reaction was rAnfirmPd by Vis l:l,V~ LU_CV,Uy. The reaction was reduced in vûlur~e to 5 mL and ~ F d on a Dowe2 50 WX-8 ion e2change column (H I form) to remove excess Ni(OAc)2 The eluent was reduced i~ volume to 5 mL and was adjusted to a pH=8.0 with 1.0 N NaOH. The resuwting solution was c~aluul~Llèv to ~5 dryness. The residue was taken up in 7 mL of MeOH and ~I..u~14gl~L,uhed on a Sephadex LH-20 column using MeOH as eluent.
Product was isolated by removal of solvent to give 0.090 g ( 85%) of the desired metAllAtL~l porphyrin as crnfir~npd by mass spectral analysis.
~,y A m~l .. 6 2~ Synth ci c of N.N ~ - ~1l . . ~r3 .3 ,1; . . ,, ",, ~ ~Al i rvli dPnpAminp) li ~ A n ~1 A ...r~.l;l;. rl: - of the ~u~,~dw~i of Coleman was used (r~oleman et al. Inorg Chem, 20, 700, [1981]). A 100 mL round bottom flask e~upped with a stir bar was charged with 25 mL of absolute EtOH
and 3 ~elLù~y~li~ladellyde ( 3.04 g, 0.0~ mol). A 20 mAL solution of 25 absolute EtOH and l.~Lyll -e l;~ P ( .601 g, 0.01 mol) was fresbly prepared and was added in one portion to the salicylaldehyde. The reaction was refiuxed for 1 h during which time a yellow-ûrange precipitate appeared. The product was collected by filtration, washed with 100 mAL of hot ethanol, and dried under vacuum to give 4.4 g (98%)0 of the desired product.
mnlP 7 Syr-~h~cic of Chlorol N.N' ~ Y~ l .;c(~ ~
dimPth~-~8Ali(`~ylill~.. ~.. ;.. AIl~)irOn (m) N,N'-Et~lylc.l~h;6(3,3'~ llelLO~alicyli~ r) ( 0.05 g, 0.188 ~5 mmole) was dwssolved in 20 r~L of MeOH and Fe(Cl)3 ( 0.030 g, 0.188 . . , ~ _ .

WO 95/31197 2 1 ~ 9 5 2 8 r~

mmole) was added in one portion. The solution was refluxed for 1 h after which time the solvent was removed under vacuum. The purple residue was washed with a minimal amount of water. The solid was taken up in 10 mL of MeOH, filtered and, c ioùlAAL~d by removal of fi solvent to give 0.047 g (70%) of the desired iron complex.
T ~ A m Pl 8 SvrlthPCiR ~If 1~ 14-DimPt~,vl-1 4 R ll-L~L~ y. ~ F . 11 .1; .AI,...;ll~l~TT) FpyAAflnlvyk~ Y Ni(TT)(rl41~iPnnN~)PFfi Ni(II)([14]dienoN4)PF6 was prepared by the method of Martin 10 and Cumming6 ( Martin, J.G.; Cummings, S.C. InnrA,. ChPm 12, 1477-1482, [1973]). The c- .,l u~ 1 was cL~ ,L~.;...;~ by mass spectral analysis and was shown to be ~- c Pl-l~l with the desired structure.
Exam~le 9 Syr thPCiR nf ~ ~ 14-DimPtllvl-1 4 R 11-I Y~ . AA . AI Y~L,L~L~ r 11 14-15 lliPnPni~kPl(TT) ~TPYAf~ ,Vh."iLhAI~ Ni(TT)(r14l~iPnPN~)(PF6~2, Ni(TT)(rl41fliPnP~l(PFfi~2 Ni(II)([14]dieneN4)(PF6)2 was prepared from Ni(II)([14]dienoN4)PF6 by the method of Martin and C ~ ( Martin, J.G.; C~mn~ir~, S.C. Innry ChPm 12, 1477-1482, [1973]).
23 E~A~le 10 SvrlthPcic ~,f fi ~ l fi 1 7-T~LI ,....~ ' l l vl~l;hpn7nrb~ilrl 4 ~ l l ltetra-I~l A~ ; 2.4 7.9.1 ~ 14-1~ YlrTT) Ni(Tlr)rl411~DnPN~
Ni(II)[14]12eneN4 was prepared by the method of Goendken et.
al. (Gopn~kpn et. al. J.C..~ ChPm Comm 337-338, [1973]). The 2fi compleA was ~ d by mass spectral analysis arld which was ' with the desired structure.
~ YmnlP 11 This esample describes the ,ul ~ Y LiOll of p.,.UA.y .L.iL~ stock solutions used in these studies. A modified version of the ~lùc~ u~t:
3D described by Hughs was used (Hughs, M. N.; Nicklin, H. G. J. Chem.
., (A), 45û452, [1968]).
To lû mL of V;~UlU~l~ly stirred 0.6 M NaNO2 solution mnintsinPd at 0 C was added an equal volume of a HCI/H2O2 solution ( 0.6 M HCI and 0.7 M H2O2) follûwed ;""~YJ;AI~ly by the rapid addition ~ . ..... .. _ _ _ _ . .

of 10 mL of 0.75 M NaOH. The resulting yellow solution was treated with 25 mg of MnO2 for 3 min. and wss . ...P l:~ h ly filtered. The filtrate was placed in a -20 C freezer for several days which resulted in the frq~ no~;~n of the sodiu~n p~.uAyl il.il~ as evident by a fine yellow 5 band visible at the top of the flask. The yellow band was collected to yield ~ 1 mL of a 280 mM sodium pc;,~,A~ liL.iL~ solution. This solution could be stirred frozen at -20 C for several dsys with rninimal ~lr ""1"'- I A.~ of ~uA.~.-iL-iL~:.
~ 1 2 10 This e~ample describes the ~u~u~e~u~O used to A~ I.P if rnmrollnrlc are p~.~A.~I~ilLil~ ~r~ :.... catalyst by stopped-flow kinetic analysis.
All analysis were run using r~ pl 7~ buffer8 (Coll ' ) which were biological grade using ultra pure water 15 prepared by the method of Riley ( Riley, D. P. et. 81. ~al. ~iQsh~m. 196, 344-349, [1991]). r~inetic ~ &~ LD were made on ar~ OLIS Rapid Scanning Stopped-Flow Sr~e Llu~ t~ ( On-Lirle LsL~ l Systems Inc., Bogart, Georgia) ) using the OLIS-RSM-1000 Operating system for data ~ uu;DiLiu~ and rn~nir~ .UA.~...I~iLP has a strong 20 ~ ... P at 302 mn ( p~tinrti~n r~rffiriPnt = 1670 M-l cm-l) and has been shown to rl- ~~ r - in a process that is first-order in sodium ~,u~l"l.iL~ ard first order in protons (IIughs, M. N.; Nicklin, H. G.
J. ChPm Snr (A), 450-452, [1968]) with tm=l.9 sec. at 37 C pH=7.4 ( Beckman, J. S. et.al. Proc. Natl. I~rs`-l Sri Ul''~. 87,1620-1624, [1990]).
25 Thus, in a tyrAical , the natural baLLKIuu~d d- (A -~ I o - rate of sodium p.,.u~.y~liL~ was A~ as follows.
A 24 mM stock solution ûf sodium ~ Ay li~ in 50 r~M NaOH is load into the small volume sy~inge and 100 rnM ~Gt~ .. rhr.-~pH=7.4 is charged into the large vAlu~ne sylinge of ~e stopped-flûw 30 D,u~..,l.,l.h ~rnPtPr All stopped -flow IIIS&DU~ 0 were ~nade at 22 C i 1 C. InJection of the sûlutions into the sample ~ ul.lya. Illlw.~
results in ~ 25 fold dilution of the stock sodium ~. uAy,~ . The dP~ ... of sodium p~ .uAy,lillile is first order in ~.uAylliLliLe with a t~ = 5.2 sec and a kob.= 1.39 x 10-1 i 0.15 secl. To test for 35 catalytic peluAyl~lri~ ~ n activity, the metal cûmplex was W095/31197 2 1 8 q 5 2 8 . ~
a7 dissolved in 100 mM p~ buffer pH=7.4 and loaded into the large syringe and the ~ rn of ~_.u~ il,-i~ was . G. i~u..,d as described above. The catalytic rate .constant (kc~t M-l sec-1) for the . ' ~ tested was ~ l by varying the complex 5 ~ --. and plotting kob~ vs [complex] Table 1. The kob~ were obtained from averages of three stopped flow analysis at each catalyst ~,v~ LnliuLL. Data .~ ~u.~ L~_ of this analysis for a variety of v ~ is shown in Figure 1. The simple di and trivalent chloride salts of Mn, Fe, Co, Cu, and Ni showed no catalytic p_.u.,~. iL-il~
10 ~lr~---,-l~o-:l . activity at CU~ iU~I of 0.050 mM and below.

-wo gs~3ll97 2 1 8 9 5 2 8 TA~LE 1 CATALYTIC RATE CONSTANTS FOR 'l~;
DECOMPOSITION OF SODIUM PEROXYNrIRlTE BY METAL
5 COMPLEXES AT pH=7.4 AND 22 C
Example No. Co~plex kcat (M-l secl) 1 Fe(III)TMPyP 2.75 x 10~6 Fe(III)TPPS a.o6 ~ 10t6 r~ )hin 320 x 10+~
OxyhPnn.~lr,hin 2.94 x 10~6 4 Fe(III)TMPS 1.60 x 10~5 5 Ni(II)TMPS 8.72 x 10~4 15 7 Fe(III)(3,3'MeO2Salen) 5.00 x 10~
3 Mn(III)TMPS 2.90 x 10~4

8 Ni(II)(tl4]dienoN4)PF6 2.05 x 10

9 Ni(II)(tl4]dieneN4)(PF6)2 1.80 ~10 Ni(II)tl4]12eneN4 1.70 x 10 2D Fe(III)EDTA 2.00 x 10~4 Fe(III)Citrste 1.50 x 10 2 H2TMPS Inactive 1 (SMP H2T~yP Inactive ZnTMPyP Inactive 2~ Ni(CR)C12A Inactive aCR=2,12~ 1 3,7,11,17-~t. ' ~lo[11.3.1]1 1(17),~1 1 ,1 ?~1 fi-pentaene b Starting material }; Y l nnlP 130 This e~ample ill~ r~tP~ the inactivatio~ of CuZn-su~u~d~
(CuZnSOD) by p~.u~y. i~iLe and that p~.u~y~lillil~
~lr . ~ ." cat~lyst shown to be active in Example 12 protect CuZnSOD again~t il~G~ Li~lGLi~ll by p~ ylliLl;Le.
Stock solutions of bovine liver CuZnSOD (DDI
35 F' ~ Inc., Mountain View CA) were prepared by ~ ~1.0 mg of enz~nne in 10 mL of 50 mM ~ c .. r~ ,.k~
buffer at a pH=7.4. The activity of this solution to dismutate superoxide O WO95/31197 21 89528 ~ ,f wa8 ~- r ; .~ ;1 by the method of Riley ( Riley, D. P. et. al. ~aL
, 196, 344-349, [1991]). All kob~ were the average of triplicate runs using a stopped flow ~ ,L~vl l ~ lu t~ - r~ I- .td by Kinetic I~L., Inc. (Ann Arbor, Ml) and was ;..1 . r-- -d to a MAC IICX
5 personal computer.
T..~ nfCll~nSODby~ ~v ;~
I~.LivdLic~ of p~oAyuilliL~ was p- ~- tl by A~ ; ~1.0 mL of stock CuZn SOD 601ution into 50 mM p: rl~n~ buffer pH=7.4 such that a final assay volume of 10 mL is achieved after

10 addition of p~. u~ liL iL~ and EDTA solutions. To these assay solution was added various amûunts of ~.u.ylliLlile ( 25 mM stock solution) such that the final ~u..~llLlALion of p_.u-ylliLIiL~ in the assay varied from 0, 25, 50, 75 and 100 uM. After the addition of y~. u.~iiii~, 100 uL of a 2.5 mM stock EDTA solution was added to each assay solution 15 such that the final oullw.~ Lion of EDTA was 250 uM. Each solution was then assayed by stopped flow analysis fûr ~uu_.u,Aid~
activity. A plot of kob, vs p~lu~ylliLIiL~ W-l~ ~.ILluLu~ is shown in Figure 2. Control reaction which cûntained CuZnSOD in the presence of 250 uM EDTA alone and 100 uM pul~F ~ nitrite or nitrate showed 2D no decrease in CuZnSOD activity.

Prot~rt.inn of C1l7.n.~0D frnnn i..~ I ~A ..I by;. IIAVII~I.I;II' lleir~
~rlu~v~.iL~ w~ nn r~tAIV8te Assay solutions were prepared as described above e~cept for 25 the addition of various of l~.u.~ML ;k~ /1 .. l.. ~:l:.. catalyst. The final solution volume was n .A . .I ~ at 10 mL. Thus, to the assay solutions Fe(m)TMPyP (0.5 and 1.0 uM final . .~ n) and Fe(m)TMPS (1.0 and 5.0 uM final c ~ -- _ rf '~i U~) was added. The solution were then treated with various amounts of p~.u~ ;L~ such 3D that the final ~ of 0,25, 50, 75 and 100 uM were achieved.
Following L.e~.~wlL with p_.u~yuiLI;~ EDTA was added to a final ~- - .- - . I ~ l: - - of 250 uM. The solutions were then assayed for SOD
activity. Plots of kob~ vs [p~.u~ylPILl;iL~] at various catalysts ~.UIlCc .lLI ulions illustrates the protective effect of Fe(m)TMPyP Figure 35 3 and Fe(m)TMPS Figure 4. Under the assay ,.. l;l:.. ,c employed, WO95/31197 21 8q528 Fe(m)TMPyP and Fe(m)TMPS were shown not to be effective catPlysts for the .l:c~ ". of ~,u,u~ ,u,idF.
~sslnU~ ^ 15 In Vitro l~vAlllp~t;~m-o M~tPr~nlc- Human re~n~hin~nt tumor necrosis factor-alpha (TNF-a) was obtained from Genzyme C~,~,u.c.Liun, Cnmhri-l~, MA. ~Iuman 1FC~ t; ,1 C5a and L-arginine (L-arg) was l,u~ s~d frûm Sigma Cher~ical Cûmpany, St. Louis, MO. A--th~n~
u~ylfiLIiL~ in 60 mM NaOH was prepared as described abûve.
10 TcnlPtinrl of F.n~ntllF~liPl CF11C Human dermal l fi~ r,s~,ular F~n~ntl~Flinl cells (HDME cells) from neonatal foreskin were prepared as ~ iuu~lly described (Marks, R.M., ~ - ' M., and Penny, R.
Tn Vit. CF~11 DevFl Biûl.. 21, 627-635 [1985~). In brief, neonatal foreskin tissue frûm several donors was washed in 70% ethanol, cut int~ small 15 pieces, then emersed in trypsin (0.6 %; Irville Scientific, Santa A~a, CA) and EDTA (1%; Sigma Chemical Company, St. Louis, MO) for 7-9 minutes. The r ~1. .l 1 .F1;A1 cells were removed by pressing the nZ. ~ d surface ûf the tissue with a scalpel blade. The cells were L~iru~ through a 35% Percoll density gradient (Sigma Cheraical 2û ~ûmpany, St. Louis, MO). After ~_..l.,; r,~ n at 250 x g fûr 10 min, cells CUIl~ /UIIV~ Ulg to a density of less than 1.048 g/ml were cûllected and plated ûnto gelatin coated tissue culture vJishes tO.1%; Sigma Chemical Company, St. Louis, MO). C ~ .".,~ cells were weeded daily using a 25 gauge needle mounted ûrlto a tuberculin syringe.
25 Purified F~n~ntllF~lip~l cells were grown to passage 5 (~8 pv~1h~
doublings) in MCDB 131 (F.n~lntllFlinl basal mevlium; Clûnetics Cul~u~liv~ with 30% human serum (BioWittaker, Inc., Walkersville, MD), 10 ng/ml EGF (Collaborative T~ F 1~ ~l Products, Bedford, MA), 2 mM L~ F (Irvine Scientific, Santa 30 Ana, CA), and 250 ~ml dibutyryl cAMP, 1 llg~ml l~l~u,,u,i,;su~e (Sir~ma Chemical Cûmpany, St. Lûuis, MO). These cells were .L~ .,4Gd as normal Pn~l~ tl F~ l cells by testing for Fntin~l-Flinl cell markers (Factor VIII h.,..~u lo.~clivi~y~ cell P . ~r~ P~nGil)tnn~in Cu~ L,lg enzyme activity, and low density li~G~. . ' uptake). Cells 35 were ~. ~v~ at passage 5 in 10% DMSO fûr use in the W095131197 2 1 8 q 5 2 8 r~l,u~ - -assays after testing negative for ~ (Coriel Institute, Camden, NJ).
PrPD~r~ti~m f N~l~t.roDhilR Human neutrophils were isolated from peripheral blood of healthy donûrs (Look, D.C., Rapp, S,R., Keller, B.T., 5 and T-T~lt~n~n, M.J. ~m J. PhYsi~l 263, L79-L87 [1992]). EDTA anti-,,ulc.lcd bloûd was separated using a single-step density ~. .1. ;r.,L".I :~ . tPMN Prep, Robbins Scientific, SuDnyvale, CA) followed by several washes in HaDk's buffered saline solution (BSS; Sigma Chemical Company, St. Louis, MO) and hypotonic Iysis ûf e.~LL~u~
10 prPr~r~t~On~ contained ~95% neutrophils and were ~95% viable by trypan blue (GIBCO T ~hrlr~t~lriP8 Grand Island NY) exclusion.
Purified neutrophils were b l~,u~ Pd in BSS 6u~ with 0.01% BSA tMiles, Inc., Rnnl'~kPP IL) and 300 uM L-arg (T-TR~RA) at a cull~c,,L.~ion of 5 x 106 cells/ml.
15 T~'n~t1 P1;~1 CP11 In;-1rV ~S: The cytoto2ic effects of Etim~ t~Pd neutrophils or p~Au..ylliilile on Pn~lAt~lPli~1 cells was rl-lP-. ;..~d using a 6lCr-release assay as described by Moldow t Moldow et. al. Methods T1'.n7:Vmr1 105, 378-385, [1984]). Passage 5 HDME cells were grOWD to a density of ~1-2 ~ 104 cell6/cmZ (~go % ",~-n,~ ) in 96 well 2~ plates and labeled for 18 h with 10 uCi/ml sodium [slCI]LL~ Lc (Amersham Cul~uul~iull, Arlington Heights, IL). The HDME cell6 were L.~LuLIl~ activated for 4 h with 100 Ulml human l.~
tumor necrosis factor-alpha (TNF-a; Genzyme C.,luulc.~iu."
Cambridge, MA), then washed twice with BSSBA- Sll~ Of 25 neutrûphils were added at a . u"~c.lL~Lull of 2.5 x 105/w~ll and allowed to settle for 15 min. Unless otherwise noted, the neutrophils were activated by priming with 25 U/ml TNF-a for 10 min fûllowed by aL~ ,ti~ with 3 llglml ' r..."~ ..t C5a (Sigma Chemical Company, St. Louis, MO). Tnrllh~t~r~ were continued for 2 h at 37 C.
30 When authentic pc.u...~ lL~l;Lc was used, it was added in the absence of neutrophils. rcLu~ylLi~liL~ was added directly to the HDME cell layer from a 25 mM stock in 50 mM NaOH giving a final ~u,l~crlL,c.Lùll from 0-800 uM. All inhibitors were made fresh ~ ' l~, prior to the assay in HBSSBA and added as 1/10 of the well volume before 35 p~i.u,~ ,iL,,~e addition or neutrûphil activation..

W0 95131197 2 1 8 9 5 21~ 32 . ~ J_ 5. ~ s ~
5lCr release was rl~ Pd by ~ of the ~
from each well (soluble fraction). The I~O~lO~ .D were washed gently with HBSSBA to remove non a&~..,L cells and the washes pooled with the soluble fraction. The adherent cells from each well.were e~ hili7pd 5 with 1 N NaOH and removed to a separate tube. Both fractions were analyzed by gamma srintillAti~-~ Du__LIu~_~lJ. Results were espressed as percent 51Cr release as follows: % release = cpm (soluble +
hu,ladL_.~.~L/ total cpm per well) x 100. Specific ~L~u;~i~;iLy reflects the difference between slCr release induced by ~ l. d neutrophils and 10 llnef;~n~ tPd neutrophils (typically 1-2% above 6r ? release).
Results were confirmed in 2-3 separate assays and the data presented are le,u~ L~L~ _.
As can be seen from Figure 5, addition of p~.u,..~,,il.:iL~ to ~ .rl~ rliAl cellg re8ults in a doge rl~ .rl- .l increase in cell ir~ury 15 d~ul DL. ,~Lllg the cytotoxic effects of p~,u~yl illiLe. Cornplexes which have been shown to be p~u~y~l;L~ .1,~ ..1 _.l: - . catalysts by stopped fiow analysis are capable of ,u~vL~_Lil.~ against p~,u~ylliLIiL~ mediated cell irUury Figure 6. These rn npl^~C are also capable of ~v~3~.LIlg against neutrophil mediated cell in,~ury in a dose A~ 1 fashion O Figure 7.
~ .YAmnle 16 protnrnl fûr Cell Prûtection A-c~vs llcin~ r~u~~ iL~ D~
~lt~lvgtc- A cell viability assay was PQtohlichpd to rapidly assess the efflcacy of p~:lu~ylliLIik(PN) catalysts in ~ u~ucLillg cells frûm PN-25 mediated iruury ard death. The p ,u~y~iLIiLe challenge consisted of apulse of synthetic PN added ~ y to cells. In order to better assess the efflcacy of PN catalysts in ,ulv~_Lillg cells from PN-mediated damage, a quantity of p~u~ylliLIik(ill 50mM NaOH) .1. '- ...;- ~1 to cause maximal inJury(100%) was added as an PYngpnnuc pulse to each 30 well of cells in the presence or absence of catalyst. The NaOH vehicle was nût toxic by itself.
Cells(lE2~AW 264.7 cells or P815 - ~Lo...& cells; American Type Culture CnllPrti~m~ Rockville, Md) were plated to ~ .n. ~ .. P on 96-well tissue culture plates. Bach well is washed twice vlith Dulbecco's 35 rh~ .h,1i.. buffered saline(DPBS; GIBCO BRL, Grand Island, NY) to remûve protein and other serum ~ which might react v~ith ~ wo 95/31197 2 1 8 9 5 2 8 the ~ c,v.y. iLlile. To each well is then added 200111 of DPBS.
PN is next placed into separate wells at increasing ~ . and cell viability l~u.- Lu~ca. The dose at which maximal cell death i8 att~ined is then utilized for the catalyst y.vti_Lv,A I e 5 PLvD,ulLALc-buffered s~line (200 uL) ~ e il.~.~ ~g cu..c~.lL.~Liuns of catalyst is ne~t placed into h viV ' -' wells of ce71s.
The maximal dose of PN is ~ n~J_~Ily 1 ~d to all wels of cell6. After 15 minutes, the medium is removed from each well and the cells are either allowed to recover overnight in Earles 10 essential medium without phenol red and ~ rl ' with 10% fetal bovine servm or alLc..l~ _ly the plate of cells is assayed that day for mitnrhnnr7riAl integrity using the Alamar Blue viability assay(Alamar P~ r-3, Inc.; nL_.~c..Lv, CA.). In either case, cells are at 37 C in 5% CO2.
15 Cell in~ury is measured as follows. Briefly, 10% Alamar Blue(v/v) in Earles MEM with 10% FBS is added to each we~ of cells for 1-2h. Ce71 "".1 A1~n1i~. . of the dye generates a nuul~3~nL product which is directly related to the number of viable cels. Moreover, the IJlv~U~iUII of the nuvlGO_c.lL ~ ~ -' is linear for over 2h. The 20 amount of nuv.~ _~i..i. product in 100111 of ~Ann~7it~nPd medium frvm each well of cels is then measured with an IDE~ nuG.~ L plate reader ( gain setting of 1%) at an emission ~ 6Lll of 575nra after exciting at 546nm. Viability is either given as absolute nuu.~a~ G.~L units or as a percent of the value obtained for untreated cells(100%).
25 As can be seen in Figure 8, both Fe- arld Ni- .iûv.,' catalysts were able to protcct the murine monocytc ~&~.v,uh6~ line RAW 264.7; in this ~ PN was added at a dose causing a 50%
decrease in cell viability.
C~ A- C~A of i~ ...,iUg PN doses on RAW and P815 cells 30 showed no evidence for a v~ ..Li~l sl~ pt;hility to ~ v.~. iL iLc-mediated injury(data not shown). However, as shown in Figure 9,there is a ~ . protection of cells by Fe-TMPyP, FeTMPS, and FeTPPS
while H2TMPyP and ZnTMPyP were relatively i..c~ Li.~ (data not shown), a result ~,n~ ,i with their lack of catalytic pûtency.
35 Addition of catalyst after PN was unable to rescue the cels from injury _ WO 95/31197 2 1 8 9 5 ~ 8 I 1/L .! - ~
3~
(data not shown) i~ the ability of the cataly6t~D to protect cells directly from o~idative damage due to PN.
~n viuo EvF~lllAti-m-5 Ç.A~ llrPd l~aw P~Pnnz The effects of p_.uAy~ iLe catalysts in uiuo were initially tested on the ~ AG~ nduced paw edema.
The choice of using this ~n uiuo ;nodel of ;..n~ ... was based on the 1~ d6~: that 1) the infl ~ response is blocked by NOS
iDhibitors and 2) by DU,U.i.UAide ,lj (SOD). Tbis irldicptes the 10 PA1 L~Lu~l of both NO and of 2 - Male Sprague Dawley rats were ~uu~ d from Harlan Sprague-Dawley (T- -1;A-.AI~A1;C IN). Male Sprague Dawley rats (175-200 g) rece*ed a r ~ . inJection in the right hind paw of l,all~6__~ (0.~ of a 1% ~ in 0.85%
saline). Paw volume was measured by a r' ' JDIllUlU_~
15 before the injection of C~.l~,__.l~l ~nd then at hourly intervals from 1 to 6 h. Edema was expressed as the increase in paw volume (in ml) measured after ~llt~ ln irljection compared to the pre-injection value for individual animals.
Rats were given a bolus i.v. injection of active or inactive 2~ ~.uA~IliLliLe catalysts 1 hour after the illL~ l&lLnl injection of Cc~ ; paw swelling was assessed thereafter every hour for up tû 6 h. ~he relative % inhibition obtained with these agents is a~ ed in Table 2. under these ~ Al r ~ c the inactive p_.~.A~ LliL~ catalysts H2TMPS, ZnTMPyP or MnTPPS (all 25 given at 30 mg/kg) or FeCI3 (5 mglkg, n=6) failed to inAhibit edema formation.
% Tnhihi1;~1n ~fPawE~lDmA byr~.u~ .iLeD~ AtA~
ISme (h) Post (~.. -~,.
3D Compound Dûse (mgA~g) 1 2 3 4 5 6 FeTMPS 3 0 g2 47 47 33 33 10 0 61 6~ 53 ~3 g7 30 0 86 aA. ~0 ~0 81 35 FeTMPy 3 0 9 10 17 6 0 woss/3lls7 2 ~ 8 q 5 2 8 FeTPPS 3 ~ iD D 19 5 lv 0 17 ~ J3 L9 ZnT~l[PS 3D 0 0 O 0 0 O
H2TMPS 3nv 0 0 0 0 0 0 MnTMPS 3n o O 0 0 0 O
10 Results are expressed as % inhihiti~n of paw edema when compared to values obtained in control rats at the same time points. Each point is the meanis.e.m for n=6 animals.
Tn~ rt;~m nf int.Pctins~l ~slm~e bv PnA~n~in in thP rat: Multiple organ15 failure syndrome (MOFS) that develops following the septic attack is in most cases fatal. The "motor" of MOFS is the ~ L,--: -~ 1 tract, in particular the small intestine. Extensive ic- ~-r ~ may be found in the intestinal mucosa due to profound v~ ~c,~ ' ;c~tinn- T~-h~Pmi~ and hypoxia result in mucous lesions, found both in animals (rat, cat, dogs) 2D and humans. The origin of the mucous lesion is hypoxia. During r. - - -. (e.g, after the initial severe v- ~r~ c~ 2- may be liberated and play an hlllJul ~u-L role in the ~ h~ C of mucous lesions in the GI tract. Intestinal damage that results from shock induced by 6l.hl..~.- 1,".- artery occlusion is p~ Lcv by bu~c~v~id~
25 ~ and LPS induced intestinal; -n~ l: - . is inhibited by non-selective in_ibitors of the nitric oxide pathway (Boughton-Smith, N.K et al., 1993). There is now ~ l and clinical evidence that suggests that excessive NO ,v.vdu~,liv.. has an l,...l r~t~ role in the l.~l,v~- ..P --~ yl.U.c, ~ to 3D V--~r~ and the cardiovascular collapse ~ Lf ~ with septic shock. Furthermore, nitric oxide synthase inhibitors prevent against the intestinal damage caused by PnAnt~.~in We have developed a model of intestinal ir~ury in rats by endotoxin and assessed the effects of IL_.c~v_~ lic al.. i- isl.~ion of ~.,.v,~. il-ile catalysts.
3v Tntr-~in~l vascv~ar pPrrnP~hilit.y was APtPrmin~d as the leakage into the jejunal tissue of [l2611-labelled bovine servm albumin ([l25n-BSA) ~ lr~ lvuDly (0.5 ml; 0.5 IlCi) together with WO95/31197 21 8~528 I~,I/L . .
either LPS (3 mgll~g, serotype 0111:B4) or isotonic saline. At 4 h after LPS a~hll3~l_Lull, segments of jejunal tissue were ligated and remûved. The intestinal tissues were rapidly washed, blotted dry and weighed. Blood (0.5 ml) was collected into tubes ~- .1- ..;..~ tri sodium 5 citrate (0.318% final cu~ Lù,l) and plasma prepared by ~_.,1 ;r,~L_I: - . (lO,OOO g ~c lO min). The {l25n-BSA corltent in segments of whole tissue and in aliquots of plasma (100 ul) was ~l. t- -:~ Fd in a gam na counter. The total content of plas~na in the intestinal tissues was e~pressed as ~l/g tissue. Changes in i l~l_V..~ ular volume in the 10 intestinal tissue was A- t~ in an -A~liti~ n_l group of rats by _.l..,:";~l~. ;"g ([125n-BSA) i~ ;.louDly 2 min before remoYal of the jejunum. The tissue and plasma content of ~ was d~ d and ill~-_v~ ular Yolume expressed as Ill/g tissue. Thi6 value was DULD~I-C~d from that obtained in the plasma lea~age studies to obtain 15 a measure of the intestinal plasma albumin leakage. After LPS
-.l...;..;-1~-1: ... (4 h), there was a ~;~DI~ ri. 1 (P<0.01) increase in the plasma leakage (from 77+10 to 224+18 ~/g tigsue, n=8). A.l . . .; . . ;~l . ,, 1 :.
of FeT~S or FeTMPyP (30 mg/l~g, i.Y, n=4), 3 h after LPS injection, caused a reduction in I ' l-~-'lF.~d albumin lealkage ~ -Fd 1 h a later, as shown in Figure lO. In contrast, ~;llis~ l of the inactive p~ .U~ylli~-i~ catalyst ZnTMPyP (30 mg/kg, i.v, n=4), 3 h after LPS injection, did not inhibit rP~in~ FllF~d albumin leakage rl~ ~....; ;~d 1 h later (Figure lO). This data was supported by hir~ Fy~minslt;rm of the jejunal tissues. When compared to 25 saline treated rats, LPS evoked profound jejunal damage with severe disruption of plicae and Yilli. LPS-induced damage was less severe in jejunums taken from rats treated with FeTMPS or FeTMPyP (30 mg/kg, i.v.).
Thus, the c ... l u ~.~7r~ which are ~ -1 u . 7C or . ' - of 3, the present invention are novel and can be utilized to treat numerous infl~.. r~ disease states and disorders. For e~ample, ._,.. r.. -:
injurY to an ischemic organ, e.g., rPrPrfilo-oA injury to the ischemic u~ liu~, in~mm~tAry bowel disease, I' ~ arthritis, GDk:OPI~ LSI Ly~ b;u~l~ psoriasis, organ ~ D~UI~I~ rejections, ,75 organ ~u~s~lv~iOn~ P radiation-induced injury, asthma, ath~ usi6, ~I.Iu.lJ)oDib, platelet aggregation, side effects from ~1~ wo 95131197 2 1 8 9 ~ 2 8 P~

drug l _...,Lu_.-L of cancer .~ infl--Pn7A, 6troke, burns, trauma, acute ~a~ ;uLLs, ~_lu.. ~,}i,iLig, hepatiti6, A~ r diseases, insulin-~lPpPnrlPnt diabetes mellitus, ~ d i- Lc,~,uD~ ular rnA~ ion, fatty Pmh-~iAm adult and infantile 5 L~, ~ Lu-.r distress, and ~ ' ~,. in neonate6.
Patients receiving IL 2 therspy often develop potentially life-LL~ L~.,iL,g side effects that include fever, chills, ll.~,u~
capillary leak syndrome, as well as evidence of multiple organ r""":. " EFPrifirr~11y including renal inc~ffiriPnry and ' -' -10 jaundice. IL-2 induces a complex network of cytokines that include tumor necrosis factor, intPr1P11kin l and 6. Therefore, IL-2-treated patients resernble patients with ~ y~u~ r~ elevated 'l'NF levels, elevated cytokine lcvels etc). Some of these induce release of free radicals as well as inducing iNOS with, ~ u-~.l release of NO.
15 A recent paper shows that iNOS is induced in patients that receive IL 2 for Ll-7~LLu_.-L of renal cell c~.. ULu~ and mA~ienAnt .. rl.. r.
(IIibbs, J.B. et al., Evidence for cytokine-inducible nitric oxide synthesis from L-argimne in patients receiving intPr1P11kin-2 therapy. J. Clin.
Invest. Vol 89, 867-877).
Activity of the C ~1~U " 1~ or r ,ul _A of the present invention for ~u~uL~Liug superoxide .1;- - -li ~ can be d~u..3..ûL_d using the stopped-fiow kinetic described above. Stopped-flow _inetic analysis is an accurate and direct method fûr ~luuu~liL~L~Iy ~ e the decay rates of ,u_.uAy. iL-iLe in water. 'lAhe stopped-flow kinetic 25 analysis is suitable fûr screening c- .rl-~- r for catalytic p_~u~ylli~liLe ~1P ~ - I :on activity and active ~ c of the present invention, as identified by stopped-flow analysis, are shown to correlate to treating the above disease states and disûrders.
In other words, the present inventiûn is for the methods and A for the LldC.LLut,ll~ of a disease or condition &d~ UC1Y
affected by ~ 0~;l " of p~lu,.~.LiL.iLe w_ich is A~ ed over a natural b~.~-uuu.d rate of decay, preferably in humans suffering from such disease or condition, which cmnrriAPA ~ a metal complex, in dosage unit form, of ~rF1 ~ 1-rate-effective 35 amounts for rlFc~ c - ~e p_.u,.y.LiL.iLe ~u~fu.u~ly wherein the metal complex is as defined above. Such methods ûr rAmrc eitiûnA

2 1 8 9 5 2B l~.,IIU",- . - ~
- , ' the l,-~I, u~i~ of these diseases without L~., lv~ y affecting norr~al 1 l-~b- lly LdY
Total daily dose ' -1---;--;-1- ~,d to a host in single or divided doses may be in amounts, for example, from about 1 to about 100 mg/kg 5 body weight daily and more usually about 3 to 30 mglkg. Dosage unit rnmrncitirnR may contain guch amounts of E--hn lll' ' - thereof to make up the daily dose. The number of ~--1 '' ,' is ,U~
about one to three times per day of about 30 mglkg per unit dosage forr~ The serum ~ c.l~luLions of the doses are about 15 ~L~ to 1.5 10 mM with preferred ranges of 3 to 300 ~
The amount of active i.~ that raay be cor~bined with the carrier materials to produce a single dosage form will vary 'iPrPn'iinE upon the host treated and the particular mode of p.l..,;,~;Fl uLion.
15 The dosage regimen for treating a d-isease condition with the C ~ -rlc and/or ~ of this irivention is selected in UIlOC with a variety.of factors, incliuding the type, age, weight, sex, diet and medical condition of the patient, the severity of the disease, the route of ~ r' ~ u~6;d~ Lullo 0 such as the activity, efficacy, rl-~ rin^~c and i ~-~,y profiles of the par~cular cn nrolln(l employed, whether a drug delivery system is utilized and whether the ~pnmrO~n~ci is ~ d as part of a drug . . ~.;..~: ... Thus, the dosage regimen actually employed may vary widely and therefore may deviate from the preferred dosage regimen 25 set forth above.
The rnnnro~~n~lc of the present invention r~ay be n~ d orally, parenterally, by inhAlAt;,~n spray, rectally, or topicaUy in dosage unit fnrm7l1At;~nc ~...,l~ ..;.,~ conventional nontoxic ~ PAlly r , ' 1 l? carriers, adjuvants, and vehicles as desired. Topical 30 ~ l.,.:,.:F~ n may also involve the use of l~ c~l~ ... 1 rr'i...;l,:~ n such as L~n~ ;1 patches or iu~Lu~hol~s;~ devices. The term parenteral as used herein includes S~ F injectirnc~
iUilU~.lUUS, intrAml~s-P~lAr~ ill~-..O'clllal in~ection, or infusion tPr~hni~lupc 35 Injectable ,UI'.~' ' ~I ""'`, for example, sterile injectable aqueous or olPAEinnlls s ~ R may be formulated according to the 4 WO 95/31197 2 1 8 9 5 2 8 r~

"nown art using suitable ~ ; e or wetting agents and b ~ e agents. T_e sterile ir~jectable preparation may also be a sterile injectable solution or E . ~ in a nontoxic parenterally r ' ' l-diluent or solvent, for example, as a solution in 1,3-'v ~l~ .. l;rl Among 5 the ~L ~ Ll_ ve~icles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are ~u,,~_..Lu,,c.lly employed as a solvent or e medium. For this purpose any Wand fixed oil may be employed including synthetic mono- or L,j;ly.~;.;d¢~. In addition, fatty lD acids such as oleic acid find use in the ,u~ Liu~l of , Ll-S l.l,c - l- ;r ~ for rectal av' . u.. . bL. of the drug can be prepared by mixing the drug with a suitable r----;- . ~ 'e excipient such as cocoa butter and polyethylene glycols which are solid at ordinary l~ " but liquid at the rectal t~lJ_.c.Lu-~ and will 15 therefore melt in the rectum and release the drug.
Solid dosage forms for or.l a l~ n may include capsules, tablets, pills, powders, granules and gels. In such solid dosage forms, the active c- . l-v .~1 may be admixed with at least one inert diluent such as sucrose lactose or starch. Such dosage forms ~I may ..180 comprise, as in nor~nal practice, nfl,l;l: .Ql s-~he~qnfPc other t~an inert diluents, e.g., 11.l.. ;. -':..~ agentg guch ag ~ '~L~ .F. ~....
stearate. In the case of capsules, tablets, and pills, the dosage forms may aiso comprise buffering agents. Tablets and pills can a~ lly be prepared with enteric coatings.
25 Liquid dosage forms for oral n~miniFf~nti~n may include ph-""n~ . Ally nl ~ Ll~ ~mlllQ ~mc golution8, QllcpPne ~nc syrups, and elixirs c- ~ inert diluents ;u u~ly used in the art, such as water. Such f -~ may also comprise adjuvants, suc~
as wetting agents, emulsifying and ~ g agents, arld fr ~s~._ t~llillg~ flavoring, and ~ ;- e agents.
VVhile the - ..l-v - l~, of the invention can be - ~ ~d as the sole active ~ ..n. ~ -l agent, they can also be used in c~hinnti~n with one or more ~- ..l u - fl~ of the present invention or with one or more c-- . l v ~ ~ w,LIiclLl are known to be effective ag~3inst 35 the specific disease state that one is targeting for ~,.,i~.,L

Claims (20)

Claims

1. A method of treating a disease in a human having such disease which is advantageously affected by the decomposition of peroxynitrite at a rate accelerated over a natural background rate of decay comprising administering a metal complex which is a peroxynitrite decomposition catalyst in unit dosage form.

2. A method of claim 1 wherein the disease is ischemic reperfusion, a side effects from drug treatment of cancer, inflammation, sepsis, stroke, multiple sclerosis or parkinson's disease..

3. A method of claim 2 wherein the disease or condition is acute or chronic inflammation.

4. A method of claim 2 wherein the disease is sepsis.

5. A method of claim 2 wherein the disease is stroke.

6. A method of claim 2 wherein the disease is ischemic reperfusion.

7. A method of claim 1 wherein the compound is a ligand structure of a metal which is selected from the group consisting of Mn, Fe, Ni and V.

8. A method of claim 7 wherein the ligand is a macrocyclic with a metal which is Mn, Fe or Ni.

9. A method of claim 4 wherein the ligand is a porphyrin or aza macrocycle containing metal.

10. A pharmaceutical composition in dosage unit form for the treatment of human diseases, including ischemic reperfusion, sepsis, chronic or acute inflammation, adult respiratory distress syndrome, cancer, bronchopulmonary dysplasia, side effects from drug treatment of cancer, cardiovascular diseases, diabetes (not included for treatment by vanadium porphyrin complexes), multiple sclerosis, colitis and specific neuronal disorders, associated with peroxynitrite comprising, per dosage unit, an amount of metal complex effective for the decomposition of peroxynitrite, said metal complex being of the formula Structure I

Z
wherein R3, R6, R9 or R12 are independently selected a group consisting of H, alkyl, alkenyl, CE2COOH. phenyl, pyridinyl, and N-alkylpyridyl such that phenyl, pyridinyl and N-alkylpyridyl are Phenyl Pyridyl N Alkylpyridine which are attached at a carbon atom, and wherein phenyl is optionally substituted by halogen, alkyl, aryl, benzyl, COOH, CONH2, SO3H, NO2, NH2, N(R)3+, NHCOR' wherein R is hydrogen, alkyl, aryl, alkaryl and R' is alkyl;
pyridinyl is optionally substituted by halogen, alkyl, aryl, benzyl, COOH CONH2, SO3H, NO2, NH2, N(R)3+, or NHCOR' wherein R and R' are as defined above; and N-alkylpyridine ring is optionally substituted by halogen alkyl, aryl, benzyl, COOH, CONH2, SO3H, NO2, NH2, N(R)3+ or NHCOR' wherein R and R' are as defined above;
R1, R2, R4, R5, R7, R9, R10, or R11 are independently selected a group consisting of H, alkyl, alkenyl, carboxyalkyl, Cl, Br, F, NO2, hydroxyalkyl, and SO3H or R1R2 can be taken together to form a ring of from 5 to 8;
X and Y are ligands or charge-neutralizing anions which are derived from any monodentate or polydentate coordinating ligand or ligand system or the corresponding anion thereof and are independently selected from the group consisting of halide, oxo, aquo, hydroxo, alcohol, phenol, dioxygen, peroxo, hydroperoxo, alkylperoxo, arylperoxo, ammonia, alkylamino, arylamino, heterocycloalkyl amino, heterocycloaryl, amino, amine oxides, hydrazine, alkyl hydrazine, aryl hydrazine, nitric oxide, cyanide, cyanate, thiocyanate, isocyanate, isothiocyarlate, alkyl nitrile, aryl nitrile, alkyl isonitrile, aryl isonitrile,nitrate, nitrite, azido, alkyl sulfonic acid, aryl sulfonic acid, alkyl sulfoxide, aryl sulfoxide, alkyl aryl sulfoxide, alkyl sulfenic acid, aryl sulfenic acid, alkyl sulfinic acid, aryl sulfinic acid, alkyl thiol carboxylic acid, aryl thiol carboxylic acid, alkyl thiol thiocarboxylic acid, aryl thiol thiocarboxylic acid, alkyl carboxylic acid, aryl carboxylic acid, urea, alkyl urea, aryl urea, alkyl aryl urea, thiourea, alkyl thiourea, aryl thiourea, alkyl aryl thiourea, sulfate, sulfite, bisulfate, bisulfite, thiosulfate, thiosulfite, hydrosulfite, alkyl phosphine, aryl phosphine, alkyl phosphine oxide, aryl phosphine oxide, alkyl aryl phosphine oxide, alkyl phosphine sulfide, aryl phosphine sulfide, alkyl aryl phosphine sulfide, alkyl phosphoric acid, aryl phosphonic acid, alkyl phosphinic acid, aryl phosphonic acid, alkyl phosphinous acid, aryl phosphinous acid, phosphate, thiophosphate, phosphite, pyrophosphite, triphosphate, hydrogen phosphate, dihydrogen phosphate, alkyl guanidino, aryl guanidino, alkyl aryl guanidino, alkyl carbamate, aryl carbamate, alkyl aryl carbamate, alkyl thiocarbamate, aryl thiocarbamate, alkyl aryl thiocarbamate, alkyl dithiocarbamate, aryl dithiocarbamate, alkyl aryl dithiocarbamate, bicarbonate, carbonate, perchlorate, chlorate, chlorite, hypochlorite, perbromate, bromate, bromite, hypobromite, tetrahalomanganate, tetrafluoroborate, hexafluorophosphate, hexafluoroanitmonate, hypophosphite, iodate, periodate, metaborate, tetraaryl borate, tetra alkyl borate, tartrate, salicylate, succinate citrate, ascorbate, saccharinate, amino acid, hydroxamic acid, thiotosylate, and anions of ion exchange resins, or systems; with the proviso that when the X and Y containing complex has a net positive charge then Z
is a counter ion which is independently X or Y, or when the X and Y containing complex has net negative charge then Z
is a counter ion selected from a group consisting of alkaline and alkaline earth cations, organic cations such as alkyl or alkylaryl ammonium cations; and M is selected from the group consisting of Mn, Fe, Ni and V;
Structure II

wherein R' is CH or N;
R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, and R16 are independently selected from a group consisting of H, SO3H, COOH, NO2, NH2, and N-alkylamino;

X, Y, Z and M are as defined above;
Structure III

A

wherein R1, R5, R9, and R13 are independently a direct bond or CH2;
R2, R2', R4, R4', R6, R6', R8, R8', R10, R10', R12, R12', R14, R14', R16, R16' are independently H, or alkyl;
R3, R7, R1, R15 are independently H or alkyl;
X, Y, Z and M are as defined above;
wherein R1, R5, R8, and R12 are independently a direct bond or CH2;
R2, R2', R4, R4', R6, R6', R7, R9, R9', R11, R11', R13, R13', R14, are independently H or alkyl;
R3 and R10 are independently H or alkyl;

X, Y, Z and M are as defined above;

wherein R1, R4, R8, R12 are independently a direct bond or CH2;
R2, R2', R3, R5, R5', R7, R9, R9', R11, R11', R13, R13', R14 are independently H or alkyl;
R10 is H or alkyl;
X, Y, Z and M are as defined above;

wherein R1, R4, R7 and R10 are independently a direct bond or CH2;
R2, R2', R3, R5, R5', R6, R8, R8', R9, R11, R11' and R12 are independently H or alkyl;
X, Y, Z and M are as defined above;

wherein R1, R4, R8 and R11 are independently a direct bond or CH2;
R2, R3, R3', R5, R5', R7, R7', R9, R10, R10', R12, R12' and R13 are independently H or alkyl;
R6 is hydrogen or alkyl;
X, Y, Z and M are as defined above;

wherein R1, R4, R7 and R10 are independently H or alkyl;
R2, R3, R3', R5, R5', R6, R8, R9, R9', R11, R11' and R12 are independently H or alkyl;
X, Y, Z and M are as defined above;

wherein R1, R3, R4 and R6 are independently H or alkyl;
R2 and 5 are independently selected from the group consisting of H, alkyl, SO3H, NO2, NH2, halogen, COOH, N(R)3+
wherein R is as defined above;
X, Y, Z and M are as defined above;

wherein R1, R2, R3, R4 are independently selected from the group consisting of H, alkyl, SO3H, NO2, NH2, halogen, COOH and N(R)3+
wherein R is as defined above;
X, Y, Z and M are as defined above;

Structure IV
Z wherein R1, R1', R2, R2', R3, R3', R4, R4', R5, R5', R6, R6', R7 and R7' are independently selected from a group consisting of H, alkyl, alkoxy, NO2, aryl, halogen, NH2, SO3H, and R6, R6', R7 and R7' may each be taken together with one other of R6, R6', R7 and R7' to form a cyclic group, preferably a 6 carbon cycloalkyl group;
M1 is Fe, Ni or V;
X, Y and Z are as defined above.

11. A method of claim 10 wherein the metal complex is structure I of the formula Z
wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, M, X, Y and Z
are as defined for Structure I in claim 10.

12. A method of Claim 10 wherein the metal complex is structure II of the formula Z wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, X, Y, M and Z are as defined for Structure II in claim 10.

13. A method of claim 10 wherein the metal complex is Structure III
A, IIIB, IIIC, IIID, IIIE, or IIIF of the formula as defined above.

14. A method of claim 10 wherein the metal complex is Structure IIIG
or IIIH of the formula as defined above.

15. A method of claim 10 wherein the metal complex is Structure IV of the formula Z

wherein R1, R1', R2 , R2', R3, R3', R4, R4', R5, R5', R6, R6', R7, R7', X, Y, Z
and M1 are as defined above.

16. A method of claim 10 wherein M is Fe.

17. A method of claim 10 wherein M is Ni.

18. A method of claim 10 wherein M is V.

19. A method of claim 10 wherein M and M1 are Mn.

20. A method of claim 11 wherein M is Fe.