Note: Descriptions are shown in the official language in which they were submitted.
woss/3lls7 r~l,o~
28
~ 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
21 89528
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
WO 95/31197 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
R11 R10
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,
R12 5M R5
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;
36
W095/31197 2 1 8 9 5 2 8
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
R8 D
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
13
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 .. ' .
21 89528 14
~L ~
R7 R7.
R6' X I R6~
R4~t ~ N=<~ ~R4-
R3~ 0-- o~R3-
R2 R1 R1- R2
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.
3J
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~
17
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
18
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~
19
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
WO95/31197 ~ 1 8~ r~ J~ -
21
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~
22
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~
23
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 ~
24
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
~ . ..... .. _ _ _ _ . .
W095/31197 21 89528
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
28
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
29
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.
14
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~ - -
31
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
10 0 13 11 28 21 2
30 0 44 43 50 32 32
FeTPPS 3 ~ iD D 19 5
lv 0 17 ~ J3 L9
0 27 25 30 34 33
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~
37
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~
39
"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
