CA2229799A1 - Bioconjugates of manganese complexes and their application as catalysts - Google Patents

Abstract

Bioconjugates of low molecular weight mimics of superoxide dismutase (SOD) represented by formula (I), wherein R, R', R1, R'1, R2, R'2, R3, R'3, R4, R'4, R5, R'5, R6, R'6, R7, R'7, R8, R'8, R9, R'9, X, Y, Z and n are as defined herein, useful as therapeutic agents for inflammatory disease states and disorders, such as ischemic/reperfusion injury, stroke, atherosclerosis, and all other conditions of oxidant-induced tissue damage or injury.

Description

W 097/06824 PCT~US96/12767 BIOCONJUGATES OF MANGANESE COMPLEXES AND THEIR
APPLICATION AS CATALYSTS

BACKGROUND OF THE INVENTION

This present invention relates to compounds effective as catalysts for dismutating superoxide. This invention relates to manganese(II) or manganese(III) complexes of nitrogen-containing fifteen-membered macrocyclic ligands which catalytically dismutate superoxide. In another aspect, this invention relates to manganese complexes of nitrogen-containing fifteen-membered macrocyclic ligands which are conjugated to a targeting biomolecule.

2. Related Art The enzyme superoxide dismutase catalyzes the conversion of superoxide into oxygen and hydrogen peroxide according to equation (1) (hereinafter referred to as dismutation). Reactive oxygen metabolites derived from superoxide are postulated to contribute to the tissue pathology in a number of ~2 ~ + ~2 ~ + 2H~ - Oz + HzOz (1) inflammatory diseases and disorders, such as reperfusion injury to the ischemic myocardium, inflammatory bowel disease, rheumatoid arthritis, osteoarthritis, atherosclerosis, hypertension, metastasis, psoriasis, organ transplant rejections, radiation-induced injury, asthma, influenza, stroke, burns and trauma. See, for example, Bulkley, G.B., Reactive oxygen metabolites and reperfusion injury: aberrant triggering of reticuloendothelial function, The Lancet, Vol. 344, pp.
934-36, October 1, 1994; Grisham, M.B., Oxidants and free radicals in inflammatory bowel disease, The Lancet, Vol. 344, pp. 859-861, September 24, 1994; Cross, C.E.
et al., Reactive oxygen species and the lung, The , WO 97/06824 PCT~US96/12767 Lancet, Vol. 344, pp. 930-33, October 1, 1994; Jenner, P., Oxidative damage in neurodegenerative disease, T~e Lancet, Vol. 344, pp. 796-798, September 17, 1994;
Cerutti, P.A., Oxy-radicals and cancer, The Lancet , Vol.
344, pp. 862-863, Sep~h~r 24, 1994 Simic, M. G., et al, Oxygen Radicals in Biology and Medicine, Basic Life Sciences, Vol. 49, Plenum Press, New York and London, 1988; Weiss J. Cell. Biochem., 1991 Suppl. 15C, 216 Abstract C110 (l991); Petkau, A., Cancer Treat. Rev. 13, 17 (1986); McCord, J. Free Radicals Biol. Med., 2, 307 (1986); and Bannister, J.V. et al, Crit. Rev. Biochem., 22, 111 (1987). The above-identified references from The Lancet teach the nexus between free radicals derived from superoxide and a variety of diseases. In particular, the Bulkley and Grisham r-eferences specifically teach that there is a nexus between the dismutation of superoxide and the final disease treatment.
It is also known that superoxide is involved in the breakdown of endothelium-derived vascular relaxing factor (EDRF), which has been identified as nitric oxide (NO), and that EDRF is protected from breakdown by superoxide dismutase. This suggests a central role for activated oxygen species derived from superoxide in the pathogenesis of vasospasm, thrombosis and atherosclerosis. See, for example, Gryglewski, R.J. et al., "Superoxide Anion is Involved in the Breakdown of Endothelium-derived Vascular Relaxing Factor", Nature, Vol. 320, pp. 454-56 (1986) and Palmer, R.M.J. et al., "Nitric Oxide Release Accounts for the Biological Activity of Endothelium Derived Relaxing Factor", Nature, Vol. 327, pp. 523-26 (1987).
Clinical trials and animal studies with natural, recombinant and modified superoxide dismutase enzymes have been completed or are ongoing to demonstrate the therapeutic efficacy of reducing superoxide levels in -CA 02229799 l99X-02-17 W O 97/06824 PCTrUS96/12767 the disease states noted above. However, numerous problems have arisen with the use of the enzymes as potential therapeutic agents, including lack of oral activity, short half-live5 in vivo, immunogenicity with nonhuman derived enzymes, and poor tissue distribution.
The manganese complexes of nitrogen-containing fifteen-membered macrocyclic ligands that are low molecular weight mimics of superoxide ~ ase (SOD) are useful as therapeutic agents and avoid many of the problems associated with SOD enzymes. However, it would be desirable to be able to direct the SOD mimics to a desired target in the body where the compound can be concentrated for optimal effect. Without some way to render the compounds "targeting", increased dosages are sometimes necessary in order to obta n an efficacious concentration at the site of interest. Such increased dosages can sometimes result in undesirable side effects in the patient.
It has now been found that the macrocycles or manganese complexes of the present invention can be attached, i.e. conjugated, to one or more targeting biomolecule(s) via a linker group to form a targeting biomolecule-macrocycle or targeting biomolecule-manganese complex conjugate.
SUMMA~Y OF THE INVENTION

It is an object of the invention to provide bioconjugates of manganese (II) or manganese (III) complexes of nitrogen-containing fifteen-membered macrocyclic ligands that are low molecular weight mimics of superoxide dismutase (SOD) which are useful as therapeutic agents for inflammatory disease states or disorders which are mediated, at least in part, by superoxide. It is a further object of the invention to provide bioconjugates of manganese (II) complexes of W O 97/06824 PCT~US96/12767 nitrogen-containing fifteen-membered macrocyclic ligands which are useful as magnetic resonance imaging (MRI) contrast agents having improved kinetic stability, improved oxidative stability and improved hydrogen bonding. It is yet a further object of the invention to provide bioconjugates of manganese complexes of nitrogen-containing fifteen-membered macrocyclic ligands that can be targeted to a specific site in the body.
According to the invention, bioconjugates of manganese (II) or manganese (III) complexes of nitrogen-containing fifteen-membered macrocyclic ligands are provided wherein (1) one to five of the "R" groups are attached to biomolecules via a linker group, (2) one of X, Y and Z is attached to a biomolecule via a linker group, or (3) one to five of the "R" ~roups and one of X, Y and Z are attached to biomolecules via a linker group; and biomolecules are independently selected from the group consisting of steroids, carbohydrates, fatty acids, amino acids, peptides, proteins, antibodies, vitamins, lipids, phospholipids, phosphates, phosphonates, nucleic acids, enzyme substrates, enzyme inhibitors and enzyme receptor substrates and the linker group is derived from a substituent attached to the "R"
group or X, Y and Z which is reactive with the biomolecule and is selected from the group consisting of -NH2, -NHRIo, -SH, -OH, -COOH, -COORlo, -CONH2, -NCO, -NCS, -cooX~, alkenyl, alkynyl, halide, tosylate, mesylate, tresylate, triflate and phenol, wherein Rlo is alkyl, aryl, or alkylaryl and Xn is a halide.
DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to bioconjugates of manganese(II) or manganese(III) complexes of nitrogen-containing fifteen-membered macrocyclic ligands which catalyze the conversion of -W O 97/06824 PCT~US96/12767 superoxide into oxygen and hydrogen peroxide. These complexes can be represented by the formula:

2 '~n ~ R3 wherein R, R', Rl, Rl', R2, R2', R3, R3', R4, R,', R5, R5', R6, R6', R7, R7', R8, R8', R9 and R9' independently represents hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylcycloalkyl, cycloalkenylalkyl, alkylcycloalkyl, alkenylcycloalkyl, alkylcycloalkenyl, alkenylcycloalkenyl, heterocyclic, aryl and aralkyl radicals and radicals attached to the ~-carbon of c~-amino acids; or Rl or R'l and R2 or R'2, R3 or R' 3 and R4 or R' 4, R5 or R'5 and R6 or R' 6 ~ R7 or R'7 and R8 or R' 8 ~ and Rg or R'9 and R or R' together with the carbon atoms to which they are attached independently form a saturated, partially saturated or unsaturated cyclic having 3 to 20 carbon atoms; or R or R' and Rl or R ~I~ R2 or R' 2 and R3 or R'3, R4 or R'4 and R5 or R' 5, R6 or R' 6 and R7 or R'7, and R8 or R'8 and R9 or R'9 together with the carbon atoms to which they are attached independently form a nitrogen containing heterocycle having 2 to 20 carbon atoms provided that when the nitrogen containing heterocycle is an aromatic heterocycle which does not contain a hydrogen attached to the nitrogen, the hydrogen attached to the nitrogen in said formula, which nitrogen is also in the macrocycle and the R groups attached to the same carbon atoms of the macrocycle are absent; and combinations thereof; and wherein (1) one to five of the "R" groups are attached to biomolecules via a linker group, (2) one of X, Y and Z is attached to a biomolecule via a linker group, or (3) one to five of the "R" groups and one of X, Y and Z are attached to biomolecules via a linker group; and biomolecules are independently selected from the group consisting of steroids, carbohydrates, fatty acids, amino acids, peptides, proteins, antibodies, VitA~i nC, lipids, phospholipids, phosphates, phosphonates, nucleic acids, enzyme substrates, enzyme inhibitors and enzyme receptor substrates and the linker group is derived from a substituent attached to the "R"
group or X, Y and Z which is reactive-with the biomolecule and is selected from the group consisting of -NH2, -NHRlo, -SH, -OH, -COOH, -COORIo, -CONH2, -NCO, -NCS, -COOXn, alkenyl, alkynyl, halide, tosylate, mesylate, tresylate, triflate and phenol, wherein Rlo is alkyl, aryl, or alkylaryl and X~ is a halide.
X, Y and Z represent suitable ligands or charge-neutralizing anions which are derived from any monodentate or polydentate coordinating ligand or ligand system or the corresponding anion thereof (for example benzoic acid or benzoate anion, phenol or phenoxide anion, alcohol or alkoxide anion). X, Y and Z 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, isothiocyanate, alkyl nitrile, aryl nitrile, alkyl isonitrile, aryl isonitrile, nitrate, nitrite, azido, alkyl sulfonic acid, aryl sulfonic acid, alkyl sulfoxide, aryl WO 97/06824 PCTrUS96/12767 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 (such as acetic acid, trifluoroacetic acid, oxalic acid), aryl carboxylic acid (such as benzoic acid, phthalic 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 phosphonic acid, aryl phosphon~c acid, alkyl phosphinic acid, aryl phosphinic 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, hexafluoroantimonate, 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 where one or more of X,Y and Z are independently attached to one or more of the "R" groups, wherein n is 0 or l. The preferred ligands from which X, Y and Z are selected include halide, organic acid, nitrate and bicarbonate anions.
The linker groups, also termed herein "linker", are derived from the specified functional groups attached to the "R" groups or X, Y and Z, and function to link the biomolecule to the "R" groups or X, Y and Z.
The functional groups are selected from the group consisting of -NH2, -NHRlo, -SH, -OH, -COOH, -COORIo~
-CONH2, -NCO, -NCS, -COOX~, alkenyl, alkynyl, halide, tosylate, mesylate, tresylate, triflate and phenol wherein Rlo is alkyl, aryl, or alkaryl and X~ is a halide. Currently, the preferred alkenyl group is ethenyl and the preferred alkynyl group is ethynyl. The functional groups on the "R" groups or X, Y and Z are reactive with the biomolecule, i.e. reactive with a functional group on the steroids, carbohydrates, fatty acids, amino acids, peptides, proteins, antibodies, Vit;~ ;n~:, lipids, phospholipids, phosphates, phosphonates, nucleic acids, enzyme substrates, enzyme inhibitors, enzyme receptor substrates and other targeting biomolecules of interest. When the functional group attached to the "R" groups or X, Y and Z reacts with the biomolecule, the functional group is modified and it is this derived functional group which is the linker. For example, when an -NH2 functional group attached to an "R" group is reacted with a steroid such as in Example 1, the linker is -NH-. The exact structure of specific linker groups will be readily apparent to those of ordinary skill in the art and will depend on the specific functional group and biomolecule selected. The specific reaction conditions for reacting a functional group attached to "R" groups or X, Y and Z
with a biomolecule will be readily apparent to those of ordinary skill in the art.
The functional group useful to form the linker, defined herein as a "linker precursor", may be present on the "R" groups at the time the macrocycle is prepared PCT~US96/12767 or it may be added or modified after preparation of the macrocycle or manganese complex thereof. Similarly, the linker precursor can be present on an axial ligand, i.e.
X, Y or Z, when the manganese complex is prepared or an exchange reaction of the axial ligands is conducted to exchange the axial ligands present in the manganese complex.
The macrocycle of the present invention can be complexed with manganese either before or after conjugation with the targeting biomolecule depending on the specific biomolecule utilized. The conjugate of the macrocyclic complex and the targeting biomolecule is defined herein as a "bioconjugate".
Targeting of drugs is well known to those of ordinary skill in the art. See, for-example, J. A.
Katzenellenbogen et al, ~ournal of Nuclear Medicine, Vol. 33, No. 4, 1992, 558, and J.A. Katzenellenbogen et al, Bioconjugate Chemistry, 1991, 2, 353.
Targeting agents are typically biomolecules. The biomolecules of the invention are biologically active molecules that are site specific, i.e. known to concentrate in the particular organ or tissue of interest. The biomolecules are selected to direct the tissue distribution of the bioconjugate via receptor binding, membrane association, membrane solubility, and the like. These biomolecules include, for example, steroids, carbohydrates (including monosaccharides, disaccharides and polysaccharides), fatty acids, amino acids, peptides, proteins, antibodies (including polyclonal and monoclonal and fragments thereof), vitamins, lipids, phospholipids, phosphates, phosphonates, nucleic acids, enzyme substrates, enzyme inhibitors and enzyme receptor substrates. The biomolecules also include those biomolecules which are combinations of the above biomolecules, such as a combination of a steroid with a carbohydrate, e.g.

digitonin.
The particular biomolecules which can be utilized to target a desired organ or tissue are known in the art or it will be readily apparent to those of ordinary skill in the art. The biomolecules of the invention are commercially available or can readily be prepared by one of ordinary skill in the art using conventional methods.
It is currently preferred that a ~-Y; ~11~ of one "R" group attached to the carbon atoms located between nitrogen atoms in the macrocycle has a biomolecule attached via a linker. In addition, the preferred compounds are those which have one to five, most preferably one to two, of the "R" groups attached to biomolecules and none of X, Y and Z attached to a biomolecule, or those which have one ~f X, Y and Z
attached to a biomolecule and none of the "R" groups attached to a biomolecule.
Currently, the preferred compounds are those wherein at least one, more preferably at least two, of the "R" groups, in addition to the "R" groups which are attached to a biomolecule, represent alkyl, cycloalkyl alkyl and aralkyl radicals and the remaining "R" groups not attached to a biomolecule represent hydrogen, a saturated, partially saturated or unsaturated cyclic or a nitrogen containing heterocycle. Other preferred groups of compounds are those wherein at least one, preferably two, of R~ or R'l and R2 or R' 2~ R3 or R'3 and R4 or R' 4, R5 or R'5 and R6 or R' 6~ R7 or R' 7 and R~ or R'", and R9 or R'9 and R or R' together with the carbon atoms to which they are attached represent a saturated, partially saturated or unsaturated cyclic having 3 to 20 carbon atoms and the remaining "R" groups in addition to the "R" groups which are attached to a biomolecule via a linker are hydrogen, nitrogen containing heterocycles or alkyl groups, and those wherein at least one, preferably two, of R or R' and R, or R~" R2 or R'2 and R3 or R'3, R4 or R' 4 and R5 or R' 5, R6 or R' 6 ~ and R~ or R'~, and R8 or R' 8 and R9 or R'9 together with the carbon atoms to which they are attached are bound to form a nitrogen containing heterocycle having 2 to 20 carbon atoms and the remaining "R" groups in addition to the "R" groups which are attached to a biomolecule via a linker are independently selected from hydrogen, saturated, partially saturated or unsaturated cyclics or alkyl groups.
As used herein, "R" groups means all of the R
groups attached to the carbon atoms of the macrocycle, R R' R R' R R' R R' R R' R R' l-e~ 2/ 2~ 3~ 3~ 4~ 4~ 5~ 5~ R6~
R'6, R~, R'~, R8, R'8, R9 and R'g.
Another embodiment of the invention is a pharmaceutical composition in unit d~sage form useful for dismutating superoxide comprising (a) a therapeutically or prophylactically effective amount of a complex as described above and (b~ a nontoxic, pharmaceutically acceptable carrier, adjuvant or vehicle.
The commonly accepted ~ch~nism of action of the manganese-based SOD enzymes involves the'cycling of the manganese center between the two oxidation states (II,III). See J. V. Bannister, W. H. Bannister, and G.
Rotilio, Crit. Rev. Biochem., 22, 111-180 (1987).

1) Mn(II) + HO2 ----> Mn(III) + HO2 2) Mn(III) + ~2 - - - - - > Mn(II) + ~2 The formal redox potentials for the ~2/~2 - and HO2/H2O2 couples at pH = 7 are -0.33 v and 0.87 v, respectively.
See A. E. G. Cass, in Metalloproteins: Part 1, Metal Proteins with Redox Roles, ed. P. Harrison, P. 121.
Verlag Chemie (Weinheim, GDR) (1985). For the above disclosed mechanism, these potentials require that a WO 97/06824 PCT~US96/12767 putative SOD catalyst be able to rapidly undergo oxidation state changes in the range of -0.33 v to 0.87 v.
The complexes derived from Mn(II) and the general class of C-substituted [15]aneN5 ligands described herein have all been characterized using cyclic voltammetry to measure their redox potential. The C-substituted complexes described herein have reversible oxidations of about +0.7 v (SHE). Coulometry shows that this oxidation is a one-electron process; namely it is the oxidation of the Mn(II) complex to the Mn(III) complex.
Thus, for these complexes to function as SOD catalysts, the Mn(III) oxidation state is involved in the catalytic cycle. This means that the Mn(III) complexes of all these ligands are e~ually competent ~s SOD catalysts, since it does not matter which form (Mn(II) or Mn(III)) is present when superoxide is present because superoxide will simply reduce Mn(III) to Mn(II) liberating oxygen.
As utilized herein, the term "alkyl", alone or in combination, means a straight-chain or branched-chain alkyl radical containing from 1 to about 22 carbon atoms, preferably from about 1 to about 18 carbon atoms, and most preferably from about 1 to about 12 carbon atoms which optionally carries one or more substituents selected from (1) -NR30R3l wherein R30 and R3l are independently selected from hydrogen, alkyl, aryl or aralkyl ; or R30is hydrogen, alkyl, aryl or aralkyl and R3l is selected from the group consisting of -NR32R33, -OH, -OR34, - C-Z, ~ bs. -g-~6, --S-~7-~d-~-t~~8)(~~s)i wherein R32 and R33 are independently hydrogen, alkyl, aryl or acyl, R34 is alkyl, aryl or alkaryl, Z is CA 02229799 l998-02-l7 hydrogen, alkyl, aryl, alkaryl, -OR34, -SR34 or -NR40R
wherein R40 and R4l are independently selected from hydrogen, alkyl, aryl or alkaryl, Z is alkyl, aryl, alkaryl, -OR34, -SR34 or -NR40R4~, R35 is alkyl, aryl, -OR34, or -NR40R4" R36 is alkyl, aryl or -NR40R4l R3, is alkyl, aryl or alkaryl, X is oxygen or sulfur, and R38 and R39 are independently selected from hydrogen, alkyl or aryl;
(2) -SR42 wherein R42is hydrogen, alkyl, aryl, alkaryl, --SR34, --NR32R33, X' o o - C -Z~ P~3, ~ -P~

wherein R43 is -OH, -OR34 or -NR32R33, and A and B are independently --OR34, -SR34 or -NR32R33 --S~~k 15 (3~

wherein x is 1 or 2, and R44 is halide, alkyl, aryl, alkaryl, --OH, --OR34, --SR34 or --NR32R33;
(4 ) -OR45 wherein R45 is hydrogen, alkyl, aryl, alkaryl, --NR32R33 ~

X' R41 0 0 --11 _Z~,--S ~~k,--P ~)~), C~r --1l ~R34)~0R~4);

wherein D and E are independently --OR34 or --NR32R33;

CA 02229799 l998-02-l7 C7-21(1~573)A

X' (5) wherein R,6 is halide, -OH, --SH, -OR3", --SR3~ or -NR3~33;
or (6) amine oxides o~ the ~ormula I R ~ 3:
o provided R30 and R3~ are not hydroqen; or t7) wherein F and G are independently --OH,--sX,--OR34, --SR34 or -NR32R3~; or (8) -O-(-(CH2)~-O)b-R~o wherein Rlo is hydrogen or alkyl, and a and b an integers independently selected ~rom 1 + 6; or (g) halo~en, cyano, nitro, or azido. Alkyl, aryl and alkaryl groups on the substituents of the above-defined alkyl groups may contain one additional substituent but are pre~erably unsubstituted. Examples o~ such radicals include methyl, ethyl, n-propyl, isopropyl, n--butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isoamyl, hexyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl and eicosyl.
The term "alkenyl", alone or in combination, means an alkyl radical having one or more double bonds. Examples o~ such alkenyl radicals include ethenyl, propenyl, 1-butenyl, cis-2-butenyl, trans-2-butenyl, iso-butylenyl, cis-2-pentenyl, trans-2-pentenyl, 3-methyl-1-butenyl, 2,3-dimethyl-2-butenyl, AM5~N~E0 SHEET
I P~/FP

CA 02229799 l998-02-l7 ~7-21 12523)A

l-pentenyl, 1-hexenyl, 1-octenyl, decenyl, dodecenyl, tetradecenyl, hexadecenyl, cis- and trans-9-octadecenyl, 1,3-pentadienyl, 2,4-pentadienyl, 2,3-pentadienyl, 1~3-hexadienyl~ 2,4-hexadienyl, 5,8,11,14-eicosatetraenyl, and 9,12,15-octadecatrienyl.
The term "alkynyl", alone or in combination, means an alkyl radical having one or more triple bonds. Examples of such alkynyl groups include ethynyl, propynyl (propar~yl), 1-butynyl, l-octynyl, 9-octadecynyl, 1,3-pentadiynyl, 2,4-pentadiynyl, 1,3-hexadiynyl, and 2,4-hexadiynyl. The term "cycloalkyl", alone or in combination means a cycloalkyl radical containing from 3 to about 10, preferably from 3 to about 8, and most preferably from 3 to about 6, carbon atoms. Examples of such cycloalkyl ~adicals include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and perhydronaphthyl. The term "cycloalkylalkyl" means an alkyl radical as defined above which is substituted by a cycloalkyl radical as defined above. Examples of cycloalkylalkyl radicals include cyclohexylmethyl, cyclopentylmethyl, (4-isopropylcyclohexyl)methyl, (4-t-butyl-cyclohexyl)methyl, 3-cyclohexylpropyl, 2-cyclo-hexylmethylpentyl, 3-cyclopentylmethylhexyl, 1-(4-neopentylcyclohexyl)methylhexyl, and 1-(4-isopropylcyclohexyl)methylheptyl. The term "cycloalkylcycloalkyl" means a cycloalkyl radical as defined above which is substituted by another cycloalkyl radical as de~ined above. Examples o~
cycloalkylcycloalkyl radicals include cyclohexylcyclopentyl and cyclohexylcyclohexyl. The term "cycloalkenyl", alone or in combination, means a cycloalkyl radical having one or more double bonds. Examples of cycloalkenyl radicals ~P'--.A'-~

CA 02229799 l998-02-l7 ~7-21tl2523)A

include cyclopentenyl, cyclohexenyl, cyclooctenyl, cyclopentadienyl, cyclohexadienyl and cyclooctadienyl. The term "cycloalkenylalkyl" means an alkyl radical as defined above which is subs.ituted by a cycloalkenyl radical as defined above. Examples of cycloalkenylalkyl radicals include 2-cyclohexen-1-ylmethyl, l-cyclopenten-}-ylmethyl, 2-(1-cyclohexen-1-yl)ethyl, 3-(1-cyclopenten-i-yl)propyl, 1-(1-cyclohexen-1-ylmethyl)pentyl, 1-(l-cyclopenten-l-yl)hexyl~
6-(1-cyclohexen-1-yl)hexyl, 1-(1-cyclopenten-1-yl?nonyl and l-(1-cyclohexen-1-yl)nonyl. The terms "alkylcycloalkyl" and "alkenylcycloalkyl" mean a cycloalkyl radical as defined above which is substituted by an alkyl or alkenyl radical as defined above.
Examples of alkylcycloalkyl and alkenylcycloalkyl radicals include 2-ethylcyclobutyl, l-~ethylcyclopentyl, 1-hexylcyclopentyl, 1-methylcyclohexyl, 1-(9-octadecenyl)cyclopentyl and 1-(9-octadecenyl)cyclohexyl. The terms "alkylcycioalkenyl" and "alkenylcycloalkenyl" means a cycloalkenyl radical as defined above which is substituted by an alkyl or alkenyl radical as defined above. Examples of alkylcycloalkenyl and alkenylcycloalkenyl radicals include 1-methyl-2-cyclopentenyl, 1-hexyl-2-cyclopentenyl, 1-ethyl-2-cyclohexenyl, 1-butyl-2-cyclohexenyl, l-(9-octadecenyl)-2-cyclohexenyl and l-(2-pentenyl)-2-cyclohexenyl. The term "aryl", alone or in combina~ion, means a phenyl or naphthyl radical which optionally carries one or more substituents selected ~rom alkyl, cycloalkyl, cycloalkenyl, aryl, heterocycle, alkoxyaryl, alkaryl, alkoxy, haloqen, hydroxy, amine, cyano, nitro, ~,,, , . , . -- ; ~ . . . ;.
. .~. _ CA 02229799 l998-02-l7 ~7-21~12523)A
-alkylthio, phenoxy, ether, tri~luoromethyl and the like, such-as phenyl, p-tolyl, 4-methoxyphenyl, 4-(tert-butoxy)phenyl, 4-fluorophenyl, 4-chlorophenyl, 4-hydroxyphenyl, 1-naphthyl, 2-naphthyl, and the like.
S The term "aralkyl", alone or in combination, means an al~yl or cycloalkyl radical as defined above in which one hydrogen atom is replaced by an aryl radical as defined above, such as benzyl, 2-phenylethyl, and the like. The term "heterocyclic" means ring structures containing at least one other kind of atom, in addition ; to carbon, in the ring. The most common of the other ~inds o~ atoms include nitroqen, oxygen and sulCur.
Examples o~ heterocyclics include pyrrolidinyl, piperidyl, imidazolidinyl, tetrahydrofuryl, tetrahydrothienyl, furyl, thienyl, pyridyl, ~uinolyl, isoquinolyl, pyridazinyl, pyrazinyl, indolyl, imidazolyl, oxazolyl, thiazolyl, pyrazolyl, pyridinyl, benzoxadiazolyl, benzothiadiazolyl, triazolyl and tetrazolyl groups. The term "saturated, partially saturated or unsaturated cyclic" means fused ring structures in which 2 carbons of the ring are also part of the fifteen-membered macrocyclic ligand. The ring structure can contain 3 to 20 carbon atoms, pre~erably 5 to lO carbon atoms, and can also contain one or more other kinds o~ atoms in addition to carbon. ~he most common of the other kinds o~ atoms include ni~roqen, oxyqen and sulfur. The ring structure can also contain more than one ring. The term "saturated, partially saturated or unsaturated ring structure" means a ring structure in which one carbon of the ring is also part of the fifteen-membered macrocyclic ligand. The rinq structure can contain 3 to 20, pre~erably S to 10, carbon atoms and can also contain nitrogen, oxygen and/or sul~ur atoms. The term "nitrogen containing heterocycle" means ring structureS in which 2 carbons and a nitrogen o~ the ring are also part of the ~i~teen-A~ SHEET
IP~A/EP

WO 97/06824 PCT~US96/12767 membered macrocyclic ligand. The ring structure can contain 2 to 20, preferably 4 to 10, carbon atoms, can be partially or fully unsaturated or saturated and can also contain nitrogen, oxygen and/or sulfur atoms in the portion of the ring which is not also part of the fifteen-membered macrocyclic ligand. The term "organic acid anion" refers to carboxylic acid anions having from about 1 to about 18 carbon atoms. The term "halide"
means chloride or bromide.
The macrocyclic ligands useful in the complexes of the present invention can be prepared according to the general procedure shown in Scheme A set forth below.
Thus, an amino acid amide, which is the corresponding amide derivative of a naturally or non-naturally occurring ~-amino acid, is reduced t~ form the corresponding substituted ethylenediamine. Such amino acid amide can be the amide derivative of any one of many well known amino acids. Preferred amino acid amides are those represented by the formula:

E~ ~ ~nH2 wherein R is derived from the D or L forms of the amino acids Alanine, Aspartic acid, Arginine, Asparagine, Cysteine, Glycine, Glutamic acid, Glutamine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Proline, Phenylalanine, Serine, Tryptophan, Threonine, Tyrosine, Valine and /or the R groups of unnatural ~-amino acids such as alkyl, ethyl, butyl, tert-butyl, cycloalkyl, phenyl, alkenyl, allyl, alkynyl, aryl, heteroaryl, polycycloalkyl, polycycloaryl, polycycloheteroaryl, imines, aminoalkyl, hydroxyalkyl, hydroxyl, phenol, amine oxides, thioalkyl, carboalkoxyalkyl, carboxylic WO 97/06824 PCT~US96/12767 acids and their derivatives, keto, ether, aldehyde, amine, nitrile, halo, thiol, sulfoxide, sulfone, sulfonic acid, sulfide, disulfide, phosphonic acid, phosphinic acid, phosphine oxides, sulfonamides, amides, amino acids, peptides, proteins, carbohydrates, nucleic acids, fatty acids, lipids, nitro, hydroxylamines, hydroxamic acids, thiocarbonyls, borates, boranes, boraza, silyl, siloxy, silaza, and combinations thereof.
Most preferred are those wherein R represents hydrogen, alkyl, cycloalkylalkyl, and aralkyl radicals. The diamine is then tosylated to produce the di-N-tosyl derivative which is reacted with a di-O-tosylated tris-N-tosylated triazaalkane diol to produce the corresponding substituted N-pentatosylpentaazacycloalkane. Th~ tosyl groups are then removed and the resulting compound is reacted with a manganese(II) compound under essentially anhydrous and anaerobic conditions to form the corresponding substituted manganese(II) pentaazacycloalkane complex.
When the ligands or charge-neutralizing anions, i.e. X, Y and Z, are anions or ligands that cannot be introduced directly from the manganese compound, the complex with those anions or ligands can be formed by conducting an exchange reaction with a complex that has been prepared by reacting the macrocycle with a manganese compound.
The complexes of the present invention, wherein R9, and R2 are alkyl, and R3, R~3, R4, R'4, R5, R'S, R6, R'6, R7, R'~, RB and R'8 can be alkyl, arylalkyl or cycloalkylalkyl and R or R~ and Rl or R~ I together with the carbon atoms they are attached to are bound to form a nitrogen containing heterocycle, can also be prepared according to the general procedure shown in Scheme B set forth below utilizing methods known in the art for preparing the manganese(II) pentaazabicyclo[12.3.1]octadecapentaene complex precursor. See, for example, Alexander et al., Inorg.

Nucl. Chem. Lett., 6, 445 (1970). Thus a 2,6-diketopyridine is condensed with triethylene tetraamine in the presence of a manganese(II) compound to produce the manganese(II) pentaazabicyclo[12.3.1]octadecapentaene complex. The manganese(II) pentaazabicyclotl2~3~l]octadecapentaene complex is hydrogenated with platinum oxide at a pressure of 10-1000 psi to give the corresponding manganese(II) pentaazabicyclotl2~3~l]octadecatriene complex.
The macrocyclic ligands useful in the complexes of the present invention can also be prepared by the diacid dichloride route shown in Scheme C set forth below. Thus, a triazaalkane is tosylated in a suitable solvent system to produce the corres~onding tris (N-tosyl) derivative. Such a derivative is treated with a suitable base to produce the corresponding disulfonamide anion. The disulfonamide anion is dialkylated with a suitable electrophile to produce a derivative of a dicarboxylic acid. This derivative of a dicarboxylic acid is treated to produce the dicarboxylic acid, which is then treated with a suitable reagent to form the diacid dichloride. The desired vicinal diamine is obtained in any of several ways. One way which is useful is the preparation from an aldehyde by reaction with cyanide in the presence of ammonium chloride followed by treatment with acid to produce the alpha ammonium nitrile. The latter compound is reduced in the presence of acid and then treated with a suitable base to produce the vicinal diamine. Condensation of the diacid dichloride with the vicinal diamine in the presence of a suitable base forms the tris(tosyl)diamide macrocycle. The tosyl groups are removed and the amides are reduced and the resulting compound is reacted with a manganese (II) compound under essentially anhydrous and anaerobic conditions to form the corresponding -substituted pentaazacycloalkane manganese (II) complex.
The vicinal diamines have been prepared by the route shown (known as the Strecker synthesis) and vicinal diamines were purchased when commercially available. Any method of vicinal diamine preparation could be used.
The macrocyclic ligands useful in the complexes of the present invention can also be prepared by the pyridine diamide route shown in Scheme D as set forth below. Thus, a polyamine, such as a tetraaza compound, containing two primary amines is condensed with dimethyl 2,6-pyridine dicarboxylate by heating in an appropriate solvent, e.g., methanol, to produce a macrocycle incorporating the pyridine ring as the 2,6-dicarboxamide. The pyridine rin~ in the macrocycle is reduced to the corresponding piperidine ring in the macrocycle, and then the diamides are reduced and the resulting compound is reacted with a manganese (II) compound under essentially anhydrous and anaerobic conditions to form the corresponding substituted pentaazacycloalkane manganese (II) complex.
The macrocyclic ligands useful in the complexes of the present invention can also be prepared by the bis(haloacetamide) route shown in Scheme E set ~orth below. Thus a triazaalkane is tosylated in a suitable solvent system to produce the corresponding tris (N-tosyl) derivative. Such a derivative is treated with a suitable base to produce the corresponding disulfonamide anion. A bis(haloacetamide), e.g., a bis(chloroacetamide), of a vicinal diamine is prepared by reaction of the diamine with an excess of haloacetyl halide, e.g., chloroacetyl chloride, in the presence of a base. The disulfonamide anion of the tris(N-tosyl) - triazaalkane is then reacted with the bis(chloroacetamide) of the diamine to produce the substituted tris(N-tosyl)diamide macrocycle. The tosyl WO 97/06824 PCT~US96/12767 groups are removed and the amides are reduced and the resulting compound is reacted with a manganese (II) compound under essentially anhydrous and anaerobic conditions to form the corresponding substituted pentaazacycloalkane manganese (II) complex.
The macrocyclic ligands useful in the complexes of the present invention, wherein R~, R l~ R2, R 2 are derived from a ~i~;no starting material and R5, R s, R"
R , and R9, R 9 can be H or any functionality previously described, can be prepared according to the pseudo-peptide method shown in Scheme F set forth below. A
substituted 1,2-diaminoethane represented by the formula gl R2 '2 H2~ ~ 2 , wherein R" R 1, R2 and R 2 are the substituents on adjacent carbon atoms in the product macrocyclic ligand as set forth above, can be used in this method in combination with any amino acids. The diamine can ~e produced by any conventional method known to those skilled in the art. The R groups in the macrocycle derived from substituents on the ~-carbon of ~-amino acids, i.e. R5, R s~ R" R 7, R9 and R 9, could be derived from the D or L forms of the amino acids Alanine, Aspartic acid, Arginine, Asparagine, Cysteine, Glycine, Glutamic acid, Glutamine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Proline, Phenylalanine, Serine, Tryptophan, Threonine, Tyrosine, Valine and/or the R groups of unnatural ~-amino acids such as alkyl, ethyl, butyl, tert-butyl, cycloalkyl, phenyl, alkenyl, allyl, alkynyl, aryl, heteroaryl, polycycloalkyl, W O 97/06824 PCT~US96/12767 polycycloaryl, polycycloheteroaryl, imines, aminoalkyl, hydroxyalkyl, hydroxyl, phenol, amine oxides, thioalkyl, carboalkoxyalkyl, carboxylic acids and their derivatives, keto, ether, aldehyde, amine, nitrile, halo, thiol, sulfoxide, sulfone, sulfonic acid, sulfide, disulfide, phosphonic acid, phosphinic acid, phosphine oxides, sulfonamides, amides, amino acids, peptides, proteins, carbohydrates, nucleic acids, fatty acids, lipids, nitro, hydroxylamines, hydroxamic acids, thiocarbonyls, borates, boranes, boraza, silyl, siloxy, silaza, and combinations thereof. As an example 1,8-dihydroxy, 4,5-diaminooctane is monotosylated and reacted with Boc anhydride to afford the differentiated N-Boc, N-tosyl derivative. The sulfonamide was alkylated with methyl bromoacetate using sodium hydride as the base and saponified to the free acid. The diamine containing N-tosylglycine serves as a dipeptide surrogate in standard solution-phase peptide synthesis.
Thus, coupling with a functionalized amino acid ester affords the corresponding pseudo-tripeptide. Two sequential TFA cleavage-couplings affords the pseudo-pentapeptide which can be N- and C-terminus deprotected in one step using HCl/AcOH. DPPA mediated cyclization followed by LiAlH4or Borane reduction affords the corresponding macrocylic ligand. This ligand system is reacted with a manganese (II) compound, such as manganese (II) chloride under essentially anaerobic conditions to form the corresponding functionalized manganese (II) pentaazacycloalkane complex. When the ligands or charge-neutralizing anions, i.e. X, Y and Z, are anions or ligands that cannot be introduced directly from the manganese compound, the complex with those anions or ligands can be formed by conducting an exchange reaction with a complex that has been prepared by reacting the macrocycle with a manganese compound.
The macrocyclic ligands useful in the complexes of the present invention, wherein Rl, R'l, R3, R'3, R5, R'5, R7, R'~, Rg and R'g can be H or any functionality as previously described, can be prepared according to the general peptide method shown in Scheme G set forth below. The R groups in the macrocycle derived from substitutents on the ~-carbon of ~-amino acids, i.e. Rl, R'l, R3, R'3, Rs~ R'5, R" R'" Rg and R'9, are defined above in Scheme F. The procedure for preparing the cyclic peptide precursors from the corresponding linear peptides are the same or significant modifications of methods known in the art. See, for example, Veber, D.F.
et al., J. Org. Chem., 44, 3101 (1979). The general method outlined in Scheme G below is an example utilizing the sequential solution-phase preparation of the functionalized linear pentapepti~e from N-terminus to C-terminus. Alternatively, the reaction sequence to prepare the linear pentapeptide can be carried out by solid-phase preparation utilizing methods known in the art. The reaction sequence could be conducted from C-terminus to N-terminus and by convergent approaches such as the coupling of di- and tri-peptides as needed.
Thus a Boc-protected amino acid is coupléd with an amino acid ester using standard peptide coupling reagents.
The new Boc-dipeptide ester is then saponified to the free acid which is coupled again to another amino acid ester. The resulting Boc-tri-peptide ester is again saponified and this method is continued until the Boc-protected pentapeptide free acid has been prepared. The Boc protecting group is removed under standard conditions and the resulting pentapeptide or salt thereof is converted to the cyclic pentapeptide. The cyclic pentapeptide is then reduced to the pentaazacyclopentadecane with lithium aluminum hydride or borane. The final ligand is then reacted with a manganese (II) compound under essentially anaerobic conditions to form the corresponding manganese (II) Wo97/06824 PCT~S96/12767 pentaazacyclopentadecane complex. When the ligands or charge-neutralizing anions, e.g. X,Y and Z, are anions or ligands that cannot be introduced directly from the manganese compound, the complex with those anions or S ligands can be formed by conducting an exchange reaction with a complex that has been prepared by reacting the macrocycle with a manganese compound.

PCT~US96/12767 O_ < R8CXE~E A ~ ~ NTs H2~ NH2N I Ts Na~

~~ .D MF
R~ R ~ R
~ 3R3 R~R'R~ 6 H2~ N~2 R ~ ~ ~ R7 TSa ~ OH Ho~R.
Tsa / Et3N

T~IIN NSIT~ ~6 OTs T

N~H /D~fF,100qC
R~

R5 R'5 Ts ~ R', /1. }3Br or H2SO~ or Na~CloHt]
/ 2. NaOH R

~N~

s R3 PCT~US96/12767 B

~R~N~

MnCI2 MeOH

F~O2 H2, 100 psi ~" MeOH, 100~C
Rg /~\

WO 97/06824 PCT~US96/12767 8C~EME C

F~R~ pTyS~ln Na N--Ts Rg--cHo d 43~cNaoc2Hs ~s ~

" 2 HCI4 BrCR2COOCH3 R 9 0~_OCH3 CH30_ ?

H N) R9 ~ H R7~ N_, T~; 2 H2. PtO2, HCI R.~S~R,R' 1. NaOH
~ ~ R ~ 2. HCI
Rg- ) ~ RR, R ~ H O H HO_ Cl HJN + +NH3 Cl ~ R7 ~~ N~,2 base ~.>~j~R, R ~ 9 ~ ~' ~ Cl Cl ~
bas~ TSR~N ~RR,2 o~o s R.~

RTS~ ,~RR2~ R9~R

~s~ R~ RR2 --R~R. ~

CH~0CH 3 renLK

h,~= \~ R~

H2, ~t~2 CH ~OH Ha R2>~ N' ~R~
~ ~ R, \ LIAIH "
\~thf ~n N RR ~,~N ' R'7 A~N~

W O 97/06824 . PCTAUS96/12767 Scheme E

R, R~~~~f~2 LW~
\ dme or thf MnC~ ~ R, CA O 2 2 2 9 7 9 9 19 9 8 0 PC~lus96ll2767 WO 97/068~4 _3 1--SC~ F s ~ ~OCE~

5 ~5 }~L~_ RL TS O R~
~L ~ R~ N ~N~ a KL;~ cP,CO~

Rg X9 ~2 ~ ~ ~0 ~ TPJ~

~>~

1~ o R5 X5 OC~3X~ ' ~I~OC~3 R3~ ~-- ~ ~C3t~O~c~ NN~--r~ ~7 8~M~ F (cont.) E~A

or BE~

MC

8C~EME G

R9 R, O EDC-HCI, HOBT, Rg R H O
>~ OH H2N Jl~ DMF, TEA, RT
BocNH ~ R >~ OEt a J~, ~ '~OEI
DMF,TEA, O ~C O R, Rl Rg R', IH o EDC-HCI, HOBT, CH30H >~N>~I~OH + H2N>:J~OEt DMF, TEA, RT
R' 'R ~' Ethyl cl '~ '~ ,.. t~"

~ ,~lEt NaOH, H20 Rs R, IH ~ R3 ~R, H O EDC-HCI, HOBT, o ~ ~ DMF, TEA, RT
eacNH ~N~ l ~N~ N~ ~OEt ~ H2N~.~ l ~
¦¦ R~ Ethyl r'~ Illdlr, /~ OEt O ~ R1 H o R S R5 DMF,TEA,0~CR 5 R5 NaOH, H20 y Rg R ~ H o R H O EDC-HCI, HOBT, >~ >~ ~ ~OH + ~OEt " Ethyl cl ,l~
O R I R1 H o R 5 R5 R, R7 DMF, TEA,0~C

W O 97/06824 PCT~US96/12767 F!M~;! G ~ cont. ) R9~?~1 NaOH. H20 CH ,OH

BocNA~ H O

~ CH2CI2 or HCI, ethet H2N>~ ~N>~ ?~N~ R j~ R~ R, o R, R1 H o R~ H O DPPA DMF TE~, R7 NH ~
o~ ~R3 LiAlH, THF. .
Ot BH3, THF

F. ~R, MDCIZ MeOH ~ f W O 97/06824 PCT~US96/12767 The pentaazamacrocycles of the present invention can possess one or more asymmetric carbon atoms and are thus capable of existing in the form of optical isomers as well as in the form of racemic or nonracemic mixtures thereof. The optical isomers can be obtained by resolution of the racemic mixtures according to conventional processes, for example by formation of diastereoisomeric salts by treatment with an optically active acid. Examples of appropriate acids are tartaric, diacetyltartaric, dibenzoyltartaric, ditoluoyltartaric and camphorsulfonic acid and then separation of the mixture of diastereoisomers by crystallization followed by liberation of the optically active bases from these salts. A different process for separation of optical isomers involves the use of a chiral chromatography column optimally chosen to ~ ; ;ze the separation of the enantiomers. Still another available method involves synthesis of covalent diastereoisomeric molecules by reacting one or more secondary amine group(s) of the compounds of the invention with an optically pure acid in an activated form or an optically pure isocyanate. The synthesized diastereoisomers can be separated by conventional means such as chromatography, distillation, crystallization or sublimation, and then hydrolyzed to deliver the enantiomerically pure ligand. The optically active compounds of the invention can likewise be obtained by utilizing optically active starting materials, such as natural amino acids.
The compounds or complexes of the present invention are novel and can be utilized to treat numerous inflammatory disease states and disorders. For example, reperfusion injury to an ischemic organ, e.g., reperfusion injury to the ischemic myocardium, surgically-induced ischemia, inflammatory bowel disease, rheumatoid arthritis, osteoarthritis, psoriasis, organ CA 02229799 l998-02-l7 ~-21(-2523)A

transplant rejections, radiation-induced injury, oxidant-induced tissue in~uries and damage, atherosclerosis, thrombosis, platelet aggregation, stroke, acute pancreatitis, insulin-dependent diabetes mellitus, disseminated intravascular coagulation, .atty embolism, adult and infantile respiratory distress, metastasis and carcinogenesis.
Activity of the compounds or complexes of the present invention for catalyzing the dismutation of superoxide can be demonstrated using the stopped-flow kinetic analysis technique as descri~ed in ~iley, D.P., Rivers, W.J. and Weiss, R.H., "Stopped-Flow Kinetic Analysis for Monitoring Superoxide Decay in Aqueous Systems," ~nal. 3iochem., 196, 344-349 (1991)~
St-opped-~low kinetic analysis is an accurate and direct method for ~uantitatively monitoring the decay rates of superoxide in water. The stopped-flow kinetic analysis is suitable ~or screening compounds ~or SOD activity and catalytic activity of the compounds or complexes o~ the present invention for dismutating superoxide, as shown by stopped-flow analysis, correlate to treating the above disease states and disorders.
Total daily dose administered to a host in single or divided doses may ~e in amounts, for example, from about l to about 100 mg/kg body weight daily and ~ore usually about 3 to 30 mg/kg. Unit dosage compositions may contain such amounts of submultiples thereof to make up the daily dose.
The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and .he particular mode of administration.
The dosage regimen ~or treating a disease condition with the compounds and/or compositions of this invention is selected in accordance with a variety of Wo97/06824 PCT~S96/12767 factors, including the type, age, weight, sex, diet and medical condition of the patient, the severity of the disease, the route of administration, pharmacological considerations such as the activity, efficacy, pharmacokinetic and toxicology profiles of the particular compound employed, whether a drug delivery system is utilized and whether the compound is a~m;ni~tered as part of a drug combination. Thus, the dosage regimen actually employed may vary widely and therefore may deviate from the preferred dosage regimen set forth above.
The compounds of the present invention may be a~ini~tered orally, parenterally, by inhAl~tion spray, rectally, or topically in dosage unit formulations containing conventional nontoxic pha~maceutically acceptable carriers, adjuvants, and vehicles as desired.
Topical administration may also involve the use of transdermal a~in;~tration such as transdermal patches or iontophoresis devices. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection, or infusion techniques.
Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in l,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In CA 02229799 l998-02-l7 ~-21(-252~)A

addition, ~atty acids such as oleic acid find use in the preparation of injectables.
Suppositories ~or rectal administration of the drug can be prepared by mixing the drug with a suitable nonirritating excipient such as cocoa butter and polyethylene glycols which are solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum and release the drug.
Solid dosage ~orms for oral administration may include capsules, tablets, pills, powders, granules and gels. In such solid dosage ~orms, the active compound may be admixed with at least one inert diluent such as sucrose lactose or starch. Such dosage forms may also comprise, as in normal practice, additional substances other than inert diluents, e.g., lubr-icating agents such as magnesium stearate. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents. Tablets and pills can additionally be pre?ared with enteric coatings.
Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water.
Such compositions may also comprise adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring, and perfuming agents.
While the compounds of the invention can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more compounds which are known to be ef~ective against the specific disease state that one is targeting for treatment.
The compounds or complexes o~ the invention can also be utilized as MRI contrast agents. A discussion of the use of contrast agents in MRI can be ~ound in patent application Serial No. 08/397,469~
A~/IENO~D S~EET
IPEA/EP

CA 02229799 l998-02-l7 07-21~12523)~
.

Contemplated equivalents of the general formulas set forth above for the compounds and derivatives as well as the intermediates are compounds otherwise corresponding thereto and having the same general properties such as tautomers of the compounds and such as wherein one or more of the various R groups are simple variations of the substituents as defined therein, e.g., wherein R is a higher alkyl group than that indicated, or where the tosyl groups are other nitrogen or oxygen protectinq groups or wherein the O-tosyl is a halide. Anions having a char~e other than 1, e.g., carbonate, phosphate, and hydrogen phos~hate, can be used instead of anions having a charge of 1, so long as they do not adversely affect-the overall activity of the complex. However, using anions having a charge other than 1 will result in a slight modification of the general ~ormula for the complex set forth above.
In addition, where a substituent is designated as, or ZO can be, a hydrogen, the exact chemical nature of a substituent which is other than hydrogen at that position, e.g., a hydrocarbyl radical or a haloqen, hydroxy, amino and the like functional group, is not critical so long as it does not adversely a~fect r he overall activity and/or synthesis procedure. Further, it is contemplated that manganese(III) complexes will be equivalent to the subject manganese(II) complexes.
The chemical reactions described above are generally disclosed in terms of their broadest application to the preparation of the compounds of this invention. Occasionally, the reactions may not be applicable as described to each compound included within the disclosed scope. The compounds ~or which this occurs will be readily recognized by those skilled in 3~ the art. In all such cases, either the reactions can be success~ully performed by conventional modifications ~MÇ~N~ D ~ ~ET

-~7-2~(125Z3)~

known to those skilled in the art, e.g., by appropriate protection o~ inter~ering groups, ~y changing to alternative conventional reagents, by routine modi~ication o~ reaction conditions, and the like, or other reactions disclosed herein or otherwise conventional, will be applicable to the preparation o~
the corresponding compounds o~ this invention. In all preparative methods, all starting materials are known or readily preparable ~rom known starting materials.

EXAMPLES

All reagents were used as received without purification unless otherwise indicated. All NMR
spectra were obtained on a Varian VXR-300 or VXR-400 nuclear magnetic resonance spectrometer. Qualitative and quantitative mass spectroscopy was run on a Finigan MATso, a Finigan 4500 and a VG40-250T using m-nitrobenzyl alcohol (NBA), m-nitrobenzyl alcohol/LiCl (NBA - Li). Melting points (mp) are uncorrected.
The ~ollowing abbreviations relatinq to amino acids and their protective groups are in accordance with the recommendation by IUPAC-IUB Commission on Biochemical Nomenclature (Biochemistry 1972, 11, 1726) and common usage.

T C

. _ , .. .

W 097/06824 PCTrUS96/12767 Ala L-Alanine DAla D-Alanine Gly Glycine Ser L-Serine 5 DSer D-Serine Bzl Benzyl Boc tert-Butoxycarbonyl Et Ethyl TFA Trifluoroacetic acid 10 DMF Dimethylformamide H0BT-H20 l-Hydroxy- (lH) -benzotriazole monohydrate EDC-HCl 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride 15 TEA Triethylamine -DMS0 Dimethylsulfoxide THF Tetrahydrofuran DPPA Diphenylphosphoryl azide *The abbreviation Cyc represents 1,2-cyclohexanediamine (stereochemistry, i.e. R,R or S,S, is indicated as such). This allows three letter code peptide nomenclature to be used in pseudopeptides containing the 1,2-cyclohexane diamine "residue".

ExamPle 1 A. Svnthesis of N-(P-toluenesulfonvl)-fR.R)-l 2-diaminocYclohexane To a stirred solution of (R,R)-1,2-diaminocyclohexane (300 g, 2.63 mole) in CH2Cl2 (5.00 1) at -10~ C was added a solution of p-toluenesulfonylchloride (209 g, 1.10 mole) in CH2Cl2 (5.00 1) dropwise over a 7 h period, maintaining the temp at -5 to -10~ C. The mixture was allowed to warm to room temp while stirring overnight. The mixture was concentrated in vacuo to a volume of 3 1 and the white _ PCT~US96/12767 solid was removed by filtration. The solution was then washed with H20 (10 x 1 1) and was dried over MgSO~.
Removal of the solvent in vacuo gave 286 g (97.5 ~
yield) of the product as a yellow crystalline solid: 'H
NMR (CDCl3) ~ 0.98 - 1.27 (m, 4 H), 1.54 - 1.66 (m, 2 H), 1.81 - 1.93 (m, 2 H), 2.34 (dt, J - 4.0, 10.7 Hz, 1 H), 2.42 (s, 3 H), 2.62 (dt, J = 4.2, 9.9 Hz, 1 H), 7.29 (d, J = 8.1 Hz, 2 H), 7.77 (d, J = 8.3 Hz, 2 H); MS
(LRFAB - DTT - DTE) m/z 269 [M + H]+.
B. SYnthesis of N-(~-toluenesulfonyl)-N -(Boc)-(R R)-1.2-diaminocvclohexane To a stirred solution of N-(p-toluenesulfonyl)-(R,R)-1,2-~; A~; nocyclohexane prepared as in Example lA
(256 g, 0.955 mole) in THF (1.15 1) was added a 1 N
solution of aqueous NaOH (1.15 1, 1.15 mole). Di-t-butyldicarbonate (229 g, 1.05 mole) was then added and the resulting mixture was stirred overnight. The layers were separated and the a~ueous layer was adjusted to pH
2 with 1 N HCl and saturated with NaCl. The aqueous solution was then extracted with CH2Cl2 (2 x 500 mL) and the extracts and THF layer were combined and dried over MgSO4. The solvent was removed in vacuo to give a yellow solid. The crude product was purified by crystallization from a THF-ether-heXanes mixture to give 310 g (88.1% yield) of the product as a white crystalline solid: mp: 137 - 139~ C; lH NMR (CDCl3) ~
1.04 - 1.28 (m, 4 H), 1.44 (s, 9 H), 1.61 - 1.69 (m, 2 H), 1.94 - 2.01 (m, 2 H), 2.43 (s, 3 H), 2.86 (brs, 1 H), 3.30 (br d, J = 9.6 Hz, 1 H), 4.37 (br d, J = 6.7 Hz, 1 H), 5.48 (br d, J = 4.6 Hz, 1 H), 7.27 (d, J = 9.7 Hz, 2 H), 7.73 (d, J = 8.1 Hz, 2 H); MS (LRFAB, NBA -Li) m/z 37S [M + Li]t.

W O 97/06824 PCT~US96/12767 C. Svnthesis of Boc-(R R)-Cvc(Ts)-qlv-OMe To a stirred solution of N-(p-toluenesulfonyl)-N -(Boc)-(R,R)-1,2-~ nocyclohexane prepared as in Example lB (310 g, 0.841 mole) in anhydrous DMF (3.11 1) at 0~ C was added NaH (37.4 g - 60 % in oil, 0.934 mole) in portions and the resulting mixture was stirred for 30 min. Methyl bromoacetate (142 g, 0.925 mole) was then added dropwise over 45 min and the mixture was allowed to warm to room temp while stirring overnight. After 10 stirring for 17 h, the solvent was removed in vacuo and the residue was dissolved in ethyl acetate(3 1) and H2O
(1 1). The ethyl acetate solution was washed with saturated NaHCO3 (1 1), saturated NaCl (500 mL) and was dried over MgSO4. The solvent was removed in vacuo and 15 the resulting oil was dissolved in ether.
Crystallization by the addition of hexanes gave 364 g (98 % yield) of the product (TLC (98:2 CHCl3--MeOH/silica gel/W detn) showed that the product contained about 5%
starting material) as colorless needles: mp of pure 20 sample lS1 - 2~ C; ~H NMR (CDCl3) ~ 1.11 - 1.22 (m, 4 H), 1.45 (s, 9 H), 1.64 -- 1.70 (m, 3 H), 2.16 -- 2.19 (m, 1 H), 2.43 (s, 3 H), 3.34 - 3.40 (m, 2 H), 3.68 (s, 3 H), 4.06 (ABq, J = 18.5 Hz, ~ = 155 Hz, 2H), 4.77 (br s 1 H), 7.30 (d, J = 8.3 Hz, 2 H), 7.82 (d, J = 8.3 Hz, 25 2 H); MS (LRFAB, DTT - DTE) m/z 441 [M + H]t.

D. Svnthesis of Boc-(R,R)--Cvc(Ts)--Glv--OH
To a stirred solution of impure Boc--(R,R)--Cyc(Ts)--Gly-OMe prepared as in Example lC (217 g, 0.492 30 mole) in MeOH (1.05 1) was slowly added a 2.5N solution of aqueous NaOH (295 mL, 0.737 mole) and the resulting solution was stirred for 2 h. The solvent was removed in vacuo and the residue was dissolved in H2O (1.5 1).
The solution was filtered to remove a small amount of 35 solid and was washed with ether (7 x 1 1) to remove the -W O 97/06824 PCT~US96/12767 impurity (compound lB) which upon drying of the combined washes over MgSO4 and removal of the solvent in vacuo resulted in recovery of 8.37 g. The pH of the aqueous solution was then adjusted to 2 with 1 N HCl and the s product was extracted with ethyl acetate (3 x 1 l).
The extracts were combined, washed with saturated NaCl (500 mL) and dried over MgSO~. The solvent was removed in vacuo and the residual ethyl acetate removed by coevaporation with ether (500 mL) and then CH2Cl2 (500 mL) to give 205 g (97.6 ~ yield) of the product as a white foam: lH NMR (CDCl3) ~ 1.15 - 1.22 (m, 4 H), 1.48 (s, 9 H), 1.55 - 1.68 (m, 3 H), 2.12 - 2.15 (m, 1 H), 2.43 (s, 3 H), 3.41 - 3.49 (m, 2 H), 3.97 (ABq, J = 17.9 Hz, A u = 69.6 Hz, 2 H), 4.79 (br s, 1 H), 7.31 (d, J =
8.1 Hz, 2 H), 7.77 (d, J = 8.3 Hz, 2-H), 8.81 (br s, 1 H); MS (LRFAB, NBA - Li) m/z 433 [M + Li]+.

. Svnthesis of Boc-(R.R)-Cyc(Ts) -G1Y - G1Y - OEt To Boc-(R,R)-Cyc(Ts)-Gly-OH (18.1 g, 43.1 mmol) in DMF (480 mL) was added HOBt-H2O (7.92 g, 51.7 mmol) and EDC-HCl (9.91 g, 51.7 mmol) and the resulting mixture was allowed to stir for 20 min at RT. To this solution was added GlyOEt-HCl (6.0 g, 43.1 mmol) and TEA
(7.2 mL, 51.7 mmol) and the resulting mixture was allowed to stir for 16 h thereafter. The DMF was evaporated and the residue was partitioned between water (250 mL) and EtOAc (400 mL). The EtOAc layer was separated and washed with lN KHSo4 (250 mL), water (250 mL), sat. NaHCO3 (250 mL) and brine (250 mL) and dried~
(Na2SO4). Filtration and concentration afforded 21.9 g (99 % yield) of pure product as a white foam: lH NMR
(DMSO-d6) ~ 1.00 - 1.10 (m, 1 H), 1.19 (t, J = 7.6 Hz, 3 H), 1.38 (s, 9 H), 1.50 - 1.56 (m, 3 H), 1.75 - 1.84 (m, 1 H), 2.38 (s, 3 H), 3.30 -3.40 (bs, 2 H), 3.75 -4.01 (complex m, 4H), 4.08 (q, J = 7.6 Hz, 2 H), 6.05 (bs, 1 _ H), 7.32 (d, J = 8.0 Hz, 2 H), 7.77 (d, J = 8.0 Hz, 2 H), 8.32 (t , J = 7.2 Hz, 1 H); MS(HRFAB) m/z 518.2551 (M + Li) t; 518.2512 calculated for C24H3~N3O~SLi.

F. Svnthesis of CYc(Ts)-GlY-GlY-OEt TFA salt To a solution of Boc-Cyc(Ts)-Gly-Gly-OEt (21.2 g, 41.4 mmol) in CH2Cl2 (180 mL) was added TFA (44 mL) and the resulting mixture was stirred at RT for 30 min. The solution was concentrated and the residue was dissolved in ether (50 mL) and precipitated with hexanes (500 mL).
The solvents were decanted and the residue was washed with 10:1 hexanes/ether (500 mL). The final residue was dried thoroughly at high vacuum to afford 20.7 g (95%
yield) of the product as a tan foam: IH NMR (DMSO-d6) ~
0.85 - 0.96 (m, 1 H), 1.03 - 1.31 (complex m, 7 H), 1.09 (t, J = 7.6 Hz, 3 H), 2.00 (m, 1 H), 2.39 (s, 3 H), 3.02 (bs, 1 H), 3.62 (m, 1 H), 3.82 - 4.05 (m, 4 H), 4.10 (q, J = 7.6, 2 H), 7.41 (d, J = 8.0 Hz, 2 H), 7.67 (d, J =
8.0 Hz, 2 H), 8.25 (bs, 3 H), 9.09 (t, J = 5.63 Hz, 1 H). MS(HRFAB) m/z 418.1990 (M -TFA + Li)+; 418.1988 calculated for Cl9H29N3O5S.

G. SYnthesis of Boc-Orn(Z)-Cvc(Ts)-Glv-Glv-OEt To Boc-Orn(Z)-OH (8.37 g, 22.8 mmol) in DMF (200 mL) was added HOBt-H2O (4.29 g, 27.4 mmol) and EDC-HCl (5.25 g, 27.4 mmol) and the resulting solution was stirred for 20 min at RT. To this solution was added Cyc(Ts)-Gly-Gly-OEt TFA salt (12.0 g, 22.8 mmol) and TEA
(3.82 mL, 27.4 mmol) and stirring was maintained for 16 h thereafter. The DMF was evaporated and the residue was partitioned between water (200 mL) and EtOAc (250 mL). The ETOAc layer was separated and washed with lN
KHSO4 (150 mL), water (150 mL), sat. NaHCO3 (150 mL) and brine (150 mL) and dried (MgSO4). Filtration and concentration afforded 15.1 g (87 % yield) of the . .

CA 02229799 l998-02-l7 W 097106824 PCT~US96/12767 product as a white foam: ~H NMR (DMSO-d6) ~ 1.00 - 1.94 (complex m, 12 H), 1.15 (t, J = 7.4 Hz, 3 H), 2.38 (s, 3 H), 2.98 (bs, 2 H), 3.30 - 3.46 (m, 2 H), 3.70 - 3.82 (m, 4 H), 3.90 ~.02 (m, 1 H), 4.05 (t, J = 7.4 Hz, 2 H), 5.00 (s, 2 H), 6.43 (m, 1 H), 7.17 (m, 1 H), 7.20 - 7.37 (m, 8 H), 7.78 (m, 2 H), 8.30 (bs, 1 H); MS(LRFAB, NBA +
HCl) m/z 760 (M + H) t-H. Svnthesis of Orn(Z)-CYc(Ts)-Glv-GlY-OEt TFA salt To a solution of Boc-Orn(Z)-Cyc(Ts)-Gly-Gly-oEt (14.5 g, 19.1 mmol) in CH2Cl2 (120 mL) was added TFA (30 mL) and the resulting solution was stirred at RT for 30 min. The solution was evaporated and the residue was triturated with ether (100 mL). The ether was decanted and the residue was dried thoroughly-at high vacuum to a~ford 15.5 g (>100 % yield, contains TFA) of the product as an orange foam: IH NMR (DMSO-d6) ~ 0.97 -1.93 (comples m, 12 H), 1.16 (t, J = 7.4 Hz, 3 H), 2.38 (s, 3 H), 2.98 (bs, 2 H), 3.31 - 3.50 (m, 2 H), 3.71 -3.91 (m, 4 H), 3.97 - 4.04 (m, 1 H), 4.08 (q, J 5 7.4 Hz, 2 H), 5.00 (s, 2H), 7.23 - 7.39 (m, 8 H), 7.77 -7.81 (m, 2H), 8.18 (bs, 3 H), 8.41 (bs, 1 H); MS(LRFAB, NBA ~ HCl) m/z 660 (M - TFA)~.

I. Svnthesis of Boc-Glv-Orn(Z)-Cvc(Ts)-Glv-Gly-OEt To a solution of Boc-Gly-OH (3.36 g, 19.2 mmol) in DMF (220 mL) was added HOBt-H2O (3.52 g, 23.0 mmol) and EDC-HCl (4.41 g, 23.0 mmol) and the resulting solution was stirred for 20 min at RT. To this solution was added Orn(Z)-Cyc(Ts)-Gly-Gly-OEt TFA salt (14.8 g, 19.2 mmol) and TEA (3.20 mL, 23.0 mmol) and stirring was maintained for 12 h thereafter. The DMF was evaporated and the residue was partitioned between water (200 mL) and EtOAc (350 mL). The layers were separated and the EtOAc layer was washed with lN KHS04 (150 mL), water (150 CA 02229799 l998-02-l7 Wo97/06824 PCT~S96/12767 -~7-mL), sat. NaHCO3 (150 mL) and brine (150 mL) and dried (MgSO4). Filtration and concentration afforded 13.7 g (87% yield) of the product as a white foam: lH NMR (DMSO-d6) ~ 0.96 - 1.10 (m, 2 H), 1.17 (t, J = 7.4 Hz , 3 H), 5 1.38 (s, 9H), 1.35 - 2.00 (complex m, lO H), 2.97 (m, 2 H), 3.60 (bs, 2 H), 3.67 - 3.84 (m, 4 H), 3.93 - 4.03 (m, 3 H), 4.06 (q, J = 7.4 Hz, 2 H), 6.92 (bs, lH), 7.19 (m, 1 H), 7.24 - 7.37 (m, 7 H), 7.60 (d, J = 8.3 Hz, 1 H), 7.76 (m, 2 H), 7.38 (bs, 1 H). MS(LRFAB, NBA + Li)+
m/z 823 (M+Li)'.

J. SYnthesis of Boc-GlY-OrnfZ)-CYc(Ts)-Glv-GlY-OH
To a solution of Boc-Gly-Orn(Z)-Cyc(Ts)-Gly-Gly-OEt (13.3 g, 16.3 mmol) in methanol (lOO mL) was added 1 15 N NaOH (25 mL). The resulting mixtufe was stirred at RT
and monitored by TLC. After 2 h the reaction was complete. The methanol was evaporated and water (50 mL) was added to the residue. This aqueous phase was washed with EtOAc ( 2 x 100 mL) and the EtOAc layers were 20 discarded. The pH was lowered to 3.5 with lN KHSO4and the aqueous phase was extracted with EtOAc ( 3 x 100 mL).
The combined EtOAc layers were dried (MgSO~), filtered and concentrated to afforded 11. 7 g (91 ~ yield) of the product as a white foam: IH NMR (CDCl3) ~ O. 98 - 1.25 25 (m, 2 H), 1.38 (s, 9 H), 1.40 - 1.92 (m, lO H), 2.38 (s, 3 H), 2.97 (m, 2 H), 3.62 (bs, 2 H), 3.75 - 3.85 (m, 3 H), 3.95 - 4.05 (m, 2 H), 5.01 (s, 2 H), 6.96 (bs, 1 H), 7.28 (m, 1 H), 7.25 - 7.38 (m, 7 H), 7.61 (d, J = 8.4 Hz, 1 H), 7.78 (m, 2 H), 8.25 (bs, 1 H).
K. Svnthesis of GlY-Orn(Z)-Cvc(Ts)-Glv-GlY-OH TFA salt ~o a solution of Boc-Gly-Orn(Z)-Cyc(Ts)-Gly-Gly-OH (11.2 g, 14.3 mmol) in CH2Cl2 (lOO mL) was added TFA
(24 mL) and the resulting solution was stirred for 30 35 min at RT. The solution was concentrated and triturated W O 97/06824 PCT~US96/12767 with ethyl ether (500 mL). Filtration of afforded 11.3 g (99 % yield) of the product as a white powder: lH NMR .
(DMSO-d6) ~ 0.95 - 1.98 (complex m, 12 H), 2.39 (s, 3 H), 3.01 (m, 2 H), 3.38 (m, 1 H), 3.65 - 4.10 (complex m, 7 H), 4.18 (q, J = 7.4 Hz, 1 H), 5.02 (s, 2 H), 7.24 -7.40 (m, 9 H), 7.77 - 7.85 (m, 2 H), 8.13 (bs, 3 H),8.31 (bs, 1 H), 8.42 (d, J = 8.3 Hz, 1 H); MS(HRFAB) 689.2953 (M-TFA)~; 689.2969 calculated for C32H45N6O9S.

L. Svnthesis of cyclo-(Glv-Orn(Z)-Cvc(Ts)-Glv-Glv-) A solution of Gly-Orn(Z)-Cvc(Ts)-Glv-GlY-OH TFA
salt (5.0 g, 6.23 mmol) in dry degassed DMF (1520 mL) was treated with TEA (1.74 mL, 12.5 mmol) and cooled to -40 ~C. DPPA (1.64 mL, 7.60 mmol was added dropwise over 10 min and the reaction was stirred at -40 ~C for 3 hr therea~ter. A~ter this time the reaction was place in a -2 ~C bath and allowed to stand at this temperature for 16 h thereafter. Water (1520 mL) was added and the resulting solution was stirred with mixed bed ion-exchange resin (750 g ) for 6 h at RT. The resin wasfiltered and the solution was concentrated to a volume of -100 mL (DMF). The addition of ethyl ether (500 mL) produced a solid residue which was redissolved in methanol (100 mL) and again precipitated by the addition of ethyl ether (500 mL). Filtration afforded 3.26 g (78 % yield) of product as a white powder: IH NMR (CDCl3) 0.96 - 2. 10 (complex m, 14 H), 2.37 (bs, 3 H), 2.68 -3.05 (m, 3 H), 3.42 - 3.90 (complex m, 8 H), 4.14 (m, 1 H), 4.20 (m, 1 H), 4.97 - 5.08 (m, 3 H), 6.42 (d, J =
8.4 Hz, 1 H), 7.19 (d, J = 8.0 Hz, 1 H), 7.20 - 7.39 (m, 7 H), 7.65 - 7.78 (m, 2 H), 9.15 (bs, 1 H), 9.22 (bs, 1 H); MS(HRFAB) m/z 671.2842 (M + H)'; 671.2863 calculated for C32H43N6O8S-W O 97/06824 PCT~US96/12767 M. Svnthesis of cYclo-(GlY-Orn-CYc(Ts)-Glv-GlY-t To a solution of cyclo-(Gly-Orn(Z)-Cyc(Ts)-Gly-Gly-) (3.94 g, 5.90 mmol) in methanol (40 mL) was added Pd (black) (1.0 g) and ammonium formate (2.0 g). The reaction was refluxed for 2 h and allowed to cool. The mixture was filtered under Argon through a pad of celite and the filtrate was concentrated to afford 2.86 g (89 %
yield) of product as a white foam: lH NMR (DMSO-d6) 0.94 - 2.22 (complex m, 12 H), 2.39 (s, 3 H), 2.55 -2.95 (m, 7 H), 3.42 - 3.89 (complex m, 9 H), 4.11 (m, 1 H), 4.39 (m, 1 H), 6,43 (d, J = 8.4 Hz, 1 H), 7.27 (d, J
- 9.3 Hz, 1 H), 7.25 - 7.45 (m, 2 H), 7.64 - 7.80 (m, 2 H), 9.12 - 9.29 (m, 2 H); MS (HRFAB) m/z 537.2511 (M +
H)'; 537.2495 calculated for C24H36N6SO6.
N. SYnthesis of cYclo-(Glv-Orn(Lithocholvl)-Cyc(Ts)-ÇlY-Gly-) To a solution of cyclo-(Gly-Orn-Cyc(Ts)-Gly-Gly-) (1.0 g, 1.9 mmol) in CHCl3 (25 mL) was added lithocholic acid NHS active ester (881 mg, 1,9 mmol) and the resulting mixture was stirred for 16 h thereafter.
Addition of ethyl ether (50 mL) produced a solid.
Filtration afforded 946 mg (56 % yield) of the product as a tan powder: 'H NMR (CD30D) ~ 0.66 (m, 3 H), 0.93 (bs, 6 H), 0.94 - 2.37 (complex m, 48 H), 2.43 (s, 3H), 2.80 - 4.60 (bm, 14 H), 7.39 (bs, 2 H), 7.80 (bs, 2 H);
MS (HRFAB) m/z 895.5432 (M + H)+; 895.5367 calculated for C~H~5N6O~S.

O. Svnthesis of 2.3-(R.R)-Cvclohexano-6-(5)-~3-(lithocholylamino)Propyl~-l 4 7 10 13-Penta-azaccloDentadecane To a suspension of cyclo-(Gly-Orn(Lithocholyl)-Cyc(Ts)-Gly-Gly-) (2.70 g, 3.00 mmol) in THF (50 mL) was added lithium aluminum hydride (51.0 mL of a 1.0 M
solution). The resulting mixture was refluxed for 16 h thereafter. The reaction mixture was cooled to --20 ~C
and ~uenched (cautiously) with 5 % Na2S04 (30 mL) followed by methanol (30 mL). This solution was stirred at RT for 1 h and concentrated to a dry powder. The powder was triturated with ethyl ether (3 x 200 mL) and filtered. The ether was concentrated and the oil was recrystallized from acetonitrile to afford 800 mg (40 %
yield) of product as a colorless oil: IH NMR (C6D6) ~
0.64 (s, 3 H), 0.67 (s, 3 H), 0.88 (d, J = 3.0 Hz, 3 H), 0.84 - 2.61 (complex m, 52 H), 2.38 - 2.95 (complex m, 14 H), 3.49 (m, 3 H); 13C NMR (CDCl3) ~ 71.4, 63.1, 62.6, 61.8, 58.2, 56.5, 56.1, 51.5, 50.4, 50.1, 48.3, 47.9, 46.1, 45.7, 42.6, 42.1, 40.4, 40.1, 36.4, 35.8, 35.7, 35.6, 35.4, 34.5, 31.9, 31.7, 31.6, 30.8, 30.5,29.4, 28.3, 27.2, 26.4, 26.2, 24.9, 24.2, ~3.4, 20.8, 18.6, 12.0; MS(LRFAB, NBA + Li~ m/z 677 (M+Li)+.

P. Synthesis of rManqanese (II) dichloro 2 3-(R.R)-CYC1 ohexano-6-(S)-~3-(lithocho 1Y1 amino)-~ro~vl~-1,4 7,10.13-~enta-azaccloDentadecane]
2,3-(R,R)-Cyclohexano-6-(S)-{3-(lithocholylamino)propyl}-1,4,7,10,13-penta-azacclopentadecane prepared as in example 10 (547 mg, 0.817 mmol) was added to a hot anhydrous methanol solution (50 mL) containing manganese (II) chloride (103 mg, 0.818 mmol) under a dry nitrogen atmosphere.
After refluxing for 2 h the solution was reduced to dryness and the residue was dissolved in a solvent mixture of THF (35 mL) and ethyl ether (5 m~) and filtered through a pad of celite. Concentration and trituration with ethyl ether afforded after filtration 512 mg (79 % yield) of the complex as a white solid: FAB
mass spectrum (NBA) m/z 760 tM-Cl]+; Anal. Calculated. !' for C41H78N60MnC12: C, 61.79; H, 9.87; N, 10.55; Cl, 8.90. Found: C, 62.67; H, 9.84; N, 8.04; Cl, 8.29.

W O 97/06824 PCT~US96/12767 Ex~mPle 2 StoPPed-Flow Kinetic AnalYsis Stopped-flow kinetic analysis has been utilized to determine whether a compound can catalyze the dismutation of superoxide (Riley, D.P., Rivers, W.J. and Weiss, R.H., "Stopped-Flow Kinetic Analysis for Monitoring Superoxide Decay in Aqueous Systems," Anal.
Biochem, 196, 344-349 [1991]). For the attainment of consistent and accurate measurements all reagents were biologically clean and metal-free. To achieve this, all buffers (Calbiochem) were biological grade, metal-free buffers and were handled with utensils which had been washed first with 0.1 N HCl, followed by purified water, followed by a rinse in a 104 M EDTA bath at pH 8, followed by a rinse with purified water and dried at 65~C for several hours. Dry DMS0 solutions of potassium superoxide (Aldrich) were prepared under a dry, inert atmosphere of argon in a Vacuum Atmospheres dry glovebox using dried glassware. The DMS0 solutions were prepared immediately before every stopped-flow experiment. A
mortar and pestle were used to grind the yellow solid potassium superoxide (-100 mg). The powder was then ground with a few drops of DMS0 and the slurry transferred to a flask containing an additional 25 ml of DMS0. The resultant slurry was stirred for 1/2 h and then filtered. This procedure gave reproducibly -2 mM
concentrations of superoxide in DMS0. These solutions were transferred to a glovebag under nitrogen in sealed vials prior to loading the syringe under nitrogen. It should be noted that the DMS0/superoxide solutions are extremely sensitive to water, heat, air, and extraneous metals. A fresh, pure solution has a very slight yellowish tint.
Water for buffer solutions was delivered from an in-house deionized water system to a Barnstead Nanopure W O 97/06824 PCT~US96/12767 Ultrapure Series 550 water system and then double distilled, first from alkaline potassium permanganate and then from a dilute EDTA solution. For example, a solution containing 1.0 g of potassium permanganate, 2 liters of water and additional sodium hydroxide n~C~ccAry to bring the pH to 9.0 were added to a 2-liter flask fitted with a solvent distillation head. This distillation will oxidize any trace of organic compounds in the water. The final distillation was carried out under nitrogen in a 2.5-liter flask cont~;n;ng 1500 ml of water from the first still and 1.0 x 106M EDTA. This step will remove remaining trace metals from the ultrapure water. To prevent EDTA mist from volatilizing over the reflux arm to the still head, the 40-cm vertical arm was packed with glass beads and wrapped with insulation. This system produces deoxygenated water that can be measured to have a conductivity of less than 2.0 nanomhos/cm2.
The stopped-flow spectrometer system was designed and manufactured by Kinetic Instruments Inc. (Ann Arbor, MI) and was interfaced to a MAC IICX personal computer.
The software for the stopped-flow analysis was provided by Kinetics Instrument Inc. and was written in QuickBasic with MacAdios drivers. Typical injector volumes (0.10 ml of buffer and 0.006 ml of DMS0) were calibrated so that a large excess of water over the DMS0 solution were mixed together. The actual ratio was approximately 19/1 so that the initial concentration of superoxide in the aqueous solution was in the range 60-120 ~M. Since the published extinction coefficient ofsuperoxide in H20 at 245 nm is -2250 Ml cm~l (1~, an initial absorbance value of approximately 0.3-0.5 would be expected for a 2-cm path length cell, and this was observed experimentally. Aqueous solutions to be mixed with the DMS0 solution of superoxide were prepared using 80 mM concentrations of the Hepes buffer, pH 8.1 (free W O 97/06824 PCT~US96/12767 acid + Na form). One of the reservoir syringes was filled with 5 ml of the DMSO solution while the other was filled with 5 ml of the aqueous buffer solution.
The entire injection block, mixer, and spectrometer cell were immersed in a thermostatted circulating water bath with a temperature of 21.0 + 0.5~C.
Prior to initiating data collection for a superoxide decay, a baseline average was obtained by injecting several shots of the buffer and DMSO solutions into the mixing chamber. These shots were averaged and stored as the baseline. The first shots to be collected during a series of runs were with aqueous solutions that did not contain catalyst. This assures that each series of trials were free of contamination capable of generating first-order superoxide deeay profiles. If the decays observed for several shots of the buffer solution were second-order, solutions of manganese(II) complexes could be utilized. In general, the potential SOD catalyst was screened over a wide range of concentrations. Since the initial concentration of superoxide upon mixing the DMSO with the aqueous buffer was -1.2 x 1o-4 M, we wanted to use a manganese (II) complex concentration that was at least 20 times less than the substrate superoxide. Consequently, we generally screened compounds for SOD activity using concentrations ranging from 5 x 10 7 to 8 x 10-6 M. Data acquired from the experiment was imported into a suitable math program (e.g., Cricket Graph) so that standard kinetic data analyses could be performed.
The catalytic rate constant for dismutation of superoxide by the manganese(II) complex of Example 1 was determined from the linear plot of observed rate constants (kob~) versus the concentration of the manganese(II) complexes. kob, values were obtained from the liner plots of ln absorbance at 245 nm versus time for the dismutation of superoxide by the manganese(II) complex. The kc~, (M~~secl) of the manganese (II) complex of Example 1 at pH c 8.1 and 21~C was determined to be 0.77 x 10'7 M-lsec-l.
The manganese(II) complex of the nitrogen-con~i n; ~g macrocyclic ligand in Example 1 is an effective catalyst for the dismutation of superoxide, as can be seen from the kc,, above.

Claims (17)

WHAT IS CLAIMED IS:

1. A compound which is a complex represented by the formula:

wherein R, R', R1, R'1, R2, R'2, R3, R'3 R4, R'4, R5, R'5, R6, R'6, R7, R'7, R8, R'8, R9 and R'9 independently represents alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylcycloalkyl, cycloalkenylalkyl, alkylcycloalkyl, alkenylcycloalkyl, alkylcycloalkenyl, alkenylcycloalkenyl, heterocyclic, aryl and aralkyl radicals and radicals attached to the .alpha.-carbon of .alpha.-amino acids; or R, or R'1 and R2 or R'2, R3 or R'3 and R4 or R'4, R5 or R'5 and R6 or R'6, R7, or R'7 and R8 or R'8, and R9 or R'9 and R or R' together with the carbon atoms to which they are attached independently form a saturated, partially saturated or unsaturated cyclic having 3 to 20 carbon atoms; or R or R' and R1 or R'6, R2 or R'2 and R3 or R'3, R4 or R'4 and R5 or R'5, R6 or R'6 and R7 or R'7, and R8 or R'8 and R9 or R'9 together with the carbon atoms to which they are attached independently form a nitrogen containing heterocycle having 2 to 20 carbon atoms provided that when the nitrogen containing heterocycle is an aromatic heterocycle which does not contain a hydrogen attached to the nitrogen, the hydrogen attached to the nitrogen in said formula, which nitrogen is also in the macrocycle and the R groups attached to the same carbon atoms of the macrocycle are absent; and combinations thereof;
wherein (1) one to five of the "R" groups are attached to biomolecules via a linker group, (2) one of X, Y and Z is attached to a biomolecule via a linker group, or (3) one to five of the "R" groups and one of X, Y and Z are attached to biomolecules via a linker group; and said biomolecules are independently selected from the group consisting of steroids, carbohydrates, fatty acids, amino acids, peptides, proteins, antibodies, vitamins, lipids, phospholipids, phosphates, phosphonates, nucleic acids, enzyme substrates, enzyme inhibitors and enzyme receptor substrates and said linker group is derived from a substituent attached to said "R" group or said X, Y and Z which is reactive with the biomolecule and is selected from the group consisting of -NH2, -NHR10, -SH, -OH, -COOH, -COOR10, -CONH2, -NCO, -NCS, -COOX", alkenyl, alkynyl, halide, tosylate, mesylate, tresylate, triflate and phenol, wherein R10 is alkyl, aryl or alkaryl and X" is a halide;
and wherein X, Y and Z are ligands 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, isothiocyanate, 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 phosphonic acid, aryl phosphonic acid, alkyl phosphinic acid, aryl phosphinic 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 thiocarbamate, alkyl dithiocarbamate, aryl dithiocarbamate, alkylaryl dithiocarbamate, bicarbonate, carbonate, perchlorate, chlorate, chlorite, hypochlorite, perbromate, bromate, bromite, hypobromite, tetrahalomanganate, tetrafluoroborate, hexafluoroantimonate, 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 the corresponding anions thereof, or X, Y and Z are independently attached to one or more of the "R" groups and n is 0 or 1.

2. Compound of Claim 1 wherein 1 to 2 of the "R" groups are attached to biomolecules via a linker group and none of X, Y and Z is attached to a biomolecule via a linker group.

3. Compound of Claim 1 wherein one of X, Y and Z is attached to a biomolecule via a linker group and none of the "R" groups are attached to biomolecules via a linker group.

4. Compound of Claim 1 wherein a maximum of one "R" group attached to the carbon atoms of the macrocycle located between nitrogen atoms has a biomolecule attached via a linker group.

5. Compound of Claim 1 wherein at least one of the "R" groups, in addition to the "R" groups which are attached to biomolecules via a linker group, are independently selected from the group consisting of alkyl, cycloalkyl, cycloalkylalkyl, aralkyl, alkary, aryl, heterocyclics and radicals attached to the .alpha.-carbon of .alpha.-amino acids, and the remaining "R" groups are independently selected from hydrogen, saturated, partially saturated or unsaturated cyclics or a nitrogen containing heterocycle.

6. Compound of Claim 5 wherein at least two of the "R" groups, in addition to the "R" groups which are attached to biomolecules via a linker group, are independently selected from the group consisting of alkyl, cycloalkyl, cycloalkylalkyl, aralkyl, alkaryl, aryl, heterocyclics and radicals attached to the .alpha.-carbon of .alpha.-amino acids.

7. Compound of Claim 5 wherein at least one of the "R" groups, in addition to the "R" groups which are attached to biomolecules, via a linker group, are alkyl and the remaining "R" groups are independently selected from hydrogen or saturated, partially saturated or unsaturated cyclics.

8. Compound of Claim 1 wherein at least one of R1 or R'1 and R2 or R'2, R3 or R'3 and R4 or R'4, R5 or R'5 and R6 or R'6, R7 or R'7 and R8 or R'8, and R9 or R'9 and R
or R' together with the carbon atoms to which they are attached represent a saturated, partially saturated or unsaturated cyclic having 3 to 20 carbon atoms and the remaining "R" groups in addition to the "R" groups which are attached to biomolecules via linker groups are independently selected from hydrogen, nitrogen containing heterocycles or alkyl groups.

9. Compound of Claim 8 wherein at least two of R1 or R'1 and R2 or R'2, R3 or R'3 and R4 or R'4, R5 or R'5 and R6 or R'6, R7 or R'7 and R8 or R'8, and R9 or R'9 and R
or R' together with the carbon atoms to which they are attached represent a saturated, partially saturated or unsaturated cyclic having 3 to 20 carbon atoms and the remaining "R" groups in addition to the "R" groups which are attached to biomolecules via linker groups are independently selected from hydrogen, nitrogen containing heterocycles or alkyl groups.

10. Compound of Claim 8 wherein said saturated, partially saturated or unsaturated cyclic is cyclohexyl.

11. Compound of Claim 10 wherein said saturated, "R" groups in addition to the "R" groups which are attached to biomolecules via linker groups are independently selected from hydrogen or alkyl groups.

12. Compound of Claim 1 wherein said R or R' and R1 or R'1, R2 or R2' and R3 or R3', R4 or R'4 and R5 or R5', R6 or R6' and R7 or R7', and R8 or R'8 and R9 or R9' together with the carbon atoms to which they are attached are found to form a nitrogen containing heterocycle having 2 to 20 carbon atoms, and the remaining "R" groups in addition to the "R" groups which are attached to biomolecules via a linker group are independently selected from hydrogen, saturated, partially saturated or unsaturated cyclics or alkyl groups.

13. Compound of Claim 1 wherein X, Y and Z are independently selected from the group consisting of halide, organic acid, nitrate and bicarbonate anions.

14. Pharmaceutical composition in unit dosage form useful for dismutating superoxide comprising (a) a therapeutically or prophylactically effective amount of a complex of Claim 1 and (b) a nontoxic, pharmaceutically acceptable carrier, adjuvant or vehicle.

15. Use of a complex of Claim 1 for preparing a medicament for preventing or treating a disease or disorder which is mediated, at least in part, by superoxide.

16. Use according to Claim 15 wherein said disease or disorder is selected from the group consisting of ischemic reperfusion injury, surgically-induced ischemia, inflammatory bowel disease, rheumatoid arthritis, atherosclerosis, thrombosis, platelet aggregation, oxidant-induced tissue injuries and damage, osteoarthritis, psoriasis, organ transplant rejections, radiation-induced injury, stroke, acute pancreatitis, insulin-dependent diabetes mellitus, adult and infantile respiratory distress, metastasis and carcinogenesis.

17. Use according to Claim 16 wherein said disease or disorder is selected from the group consisting of ischemic reperfusion injury, stroke, atherosclerosis and inflammatory bowel disease.