CZ271198A3 - Manganese bioconjugates of iron complexes of macrocyclic ligands containing nitrogen, acting as catalysts for superoxide dismutation - 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, M 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

Field of technology

The invention relates to compounds acting as catalysts for superoxide dismutation. The invention relates to manganese or iron complexes of fifteen-membered nitrogen-containing macrocyclic ligands that catalytically dismute superoxide. In another aspect, the invention relates to manganese or iron complexes of fifteen-membered nitrogen-containing macrocyclic ligands that are conjugated to a target biomolecule.

Current state of the art ..... ....... .....................

The enzyme superoxide dismutase catalyzes the conversion of superoxide into oxygen and hydrogen peroxide according to equation (1) (referred to here as dismutation).

O2 + O2 ~ + 2H —> O2 + H2O2 iU of reactive oxygen metabolites derived from superoxide, their participation in the pathological process of the tissue in a number of inflammatory diseases and disorders, such as reperfusion injury of the ischemic myocardium, inflammatory bowel disease, rheumatoid arthritis, osteoarthritis, atherosclerosis, hypertension, metastases, psoriasis, rejection of transplanted organs, damage caused by radioactive radiation, 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, pages 934-936, 1, October, 1994; Grisham, M. B., Oxidants and free radicals in inflammatory bowel disease, The Lancet, vol. 344, page 859-861, October 24, 1994; Cross, C.E. et al., Reactive oxygen species and the lung, The Lancet, vol. 344, page 930-933, October 1, 1944; Jenner P., Oxidative damage in neurodegenerative disease, The Lancet, vol. 344, page 796-798, October 17, 1944; Ceruttí, P. A., Oxy-radicals and cancer, The Lancet, vol. 344, page 862-863, October 24, 1994; Simic, M.G., et al., Oxygen Radicals in Biology and Medicine, Basic Life Science, Vol. 49, Plenum Press, New York and London, 1988; Weiss J. Cell. Biochem., 1991 Suppl. 15C, 216 Abstract C110 (1991); Petkau, A., Cancer Treat. Roar. 13, 17 (1986); McCord, J. Free Radicals Biol. Μβά.,_2^ 307 (1986); and Bannister, J.V. et al., Crit. Roar. Biochem., 22, 111, (1987). The references from The Lancet above discuss * in ,« « « * ». » » ·

99999« · 9 9 ··· · 9

9 9 9 9 » 9 9 • 9 9 »·· 9 99 ·9 99 -y ο connections between free radicals derived from superoxide and various diseases. More specifically, the Bulkley and Grisham references directly discuss the link between superoxide dismutation and the ultimate treatment of disease.

Superoxide is also known to play a role in the impairment of endothelin-derived vascular relaxing factor (EDRF), which has been identified as nitric oxide (NO), and that EDRF is protected from degradation by superoxide dismutase. This fact assumes a major role of activated oxygen particles derived from superoxide in the pathogenesis of vasospasm, thrombosis and atherosclerosis. See, for example, Gryglewski, RJ et al., Superoxide Anion is Involved in the Breakdown of Endothelium-derived Vascular Relaxing Factof ¹ , Nature, vol. 320, pp. 454-456 (1986) and Palmer, RMJ et al., "Nitric Oxide Release Accounts for the Biological Activity of Endothelium Derived Relaxing Factor", Nature, vol. 327, pp. 523-526 (1987). ...... ................

Clinical successes and animal studies with natural, recombinant and modified superoxide dismutase enzymes; which have been completed or are ongoing, demonstrate the therapeutic effect of reduced superoxide levels in the disease states listed above. However, many problems have arisen with the use of enzymes as potential therapeutic agents, including lack of activity for oral administration, short in vivo half-life, immunogenicity of non-human-derived enzymes, and poor tissue distribution.

Manganese or iron complexes of fifteen-membered nitrogen-containing ligands, which have a low molecular weight, mimic the function of superoxide dismutase (SOĎ) and are useful as therapeutic substances. This eliminates many of the problems associated with SOD enzymes. What is desired is the ability to precisely mimic SOD for the desired effect in the body where the compound can be concentrated. For non-targeted compounds, increased doses are sometimes necessary to achieve an effective concentration at the desired site. These increased doses can sometimes result in adverse effects in the patient.

It has now been found that the manganese or iron macrocycles or complexes of the invention can be attached, i.e. conjugated, to one or more target molecule(s) via a linking group to form a target biomolecule macrocycle or target biomolecule-manganese complex conjugate.

r »44 4 · ·· ·· · · · · · • ·4· 44 4

4 4 · • 4 4 4 4 • 4 4 . 44 4 4

The essence of the invention

The subject of the invention are new bioconjugates of manganese or iron complexes of fifteen-membered nitrogen-containing macrocyclic ligands, which have a low molecular weight, mimic the function of superoxide dismutase (SOD), and are useful as therapeutic substances in inflammatory disease states or disorders that are caused, at least in part, by superoxide . Another object of the invention are new bioconjugates of manganese or iron complexes of fifteen-membered macrocyclic ligands containing nitrogen, which can be used as contrast agents in magnetic resonance imaging (MRI) with higher kinetic stability and better hydrogen bonding. Another object of the invention are bioconjugates of manganese or iron complexes of fifteen-membered nitrogen-containing macrocyclic ligands, which can be targeted to a specific place in the body.

According to the invention, bioconjugates of manganese or iron complexes of fifteen-membered nitrogen-containing macrocyclic ligands are formed in such a way that (1) one to five R” groups are attached to the biomolecule by means of a linking group, (2) X, Y or Z is attached to the biomolecule by means of a linking group , or (3) one to five ``R'' groups and one of X, Y, or Z are attached to the biomolecule via a linking group; and the biomolecules are independently selected from the group consisting of steroids, bicarbonates, fatty acids, amino acids, peptides, proteins, antibodies, vitamins, lipids, phospholipids, phosphates, phosphonates, nucleic acids, enzyme substrates, enzyme inhibitors, and enzyme receptor substrates, and a binding group is derived from a substituent attached to the R” group or X, Y and Z that is reactive in the biomolecule and is selected from the group consisting of -NH ₂ , -NHR10, -SH, -OH, -CÓÓH, -COOR10, CONH ₂ , -NCO, -NCS, -COOX”, alkenyl, alkynyl, halide, p-toluenesulfonate, methanesulfonate, tresylate, triflate and phenol, where Rw is alkyl, aryI, or alkylaryI and X” is halide.

The invention is focused on bioconjugates of manganese or iron complexes of fifteen-membered nitrogen-containing macrocyclic ligands, which catalyze the conversion of superoxide into oxygen and hydrogen peroxide. These complexes are represented by the general formula I:

• 4 » 4 4« • 4 44*4 « 4 « »44 • 44*4 · • 4 4 4

44 44

where R, R', R1, R/. R2, R2, R3, R3, R4. R4, Rs, Rs, Re. Re, R7, _R7 , Re, Rs, Rg and _Rg are independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylcycloalkyl, cycloalkenylalkyl. alkylcycloalkyl, alkenylcycloalkyl, alkylcycloalkenyl, alkenylcycloalkenyl, heterocyclic, aryl and aralkyl radicals and radicals attached to the α-carbon of an α-amino acid; or R1 or R/ and R ₂ or R ₂ , R3 or R3 and R4 or R4 , Rs or Rg and R ₆ or R ₆ , R ₇ or R ₇ and Re or Re and Rg or Rg and R or R together with carbon by the atom to which they are attached they form an independently saturated, partially saturated or unsaturated ring of 3 to 20 carbon atoms; or R or R and Rj or Ri and R ₂ or R ₂ , R 3 or R 3 and R 4 or R 4 , R ₅ or R ₅ and Re or R 6 , R ₇ or R ₇ and R ₈ or Re and Rg or Rg and R or R together with the carbon atom to which they are attached independently form a nitrogen-containing heterocycle of 2 to 20 carbon atoms, provided that when the nitrogen-containing heterocycle is an aromatic heterocycle that does not contain a hydrogen attached to the nitrogen, the hydrogen attached to the nitrogen of said formula, this nitrogen is also in the macrocycle and the R group attached to the same carbon atom of the macrocycle is not present; and combinations thereof; and wherein (1) one to five "R" groups are attached to the biomolecule via a linking group, (2) one of X, Y, and Z is attached to the biomolecule via a linking group, or (3) one to five "R" groups and one of X, Y and Z is attached to the biomolecule by means of a linking group; and the biomolecules are independently selected from the group consisting of steroids, bicarbonates, fatty acids, amino acids, peptides, proteins, antibodies, vitamins, lipids, phospholipids, phosphates, phosphonates, nucleic acids, enzyme substrates, enzyme inhibitors, and enzyme receptor substrates, and a binding group is derived from a substituent attached to the "R" group or X, Y and Z that is reactive with the biomolecule and is selected from the group consisting of -NH _2l -NHR ₁₀ , -SH, -OH, -COOH, -COOR10, - CONH ₂₎ -NCO, 5 · fc · · ·· • » ·♦·· · · · · *·· » * *«· · * · · · *« * ··» «»· *« ·ι

NCS, -COOX, alkenyl, alkynyl, halide, p-toluenesulfonate, methanesulfonate, tresylate, triflate, and phenol, wherein R ₁₀ is alkyl, aryl, or alkylaryl and X" is a halide; and where M is Mn or Fe.

X, Y and Z represent suitable ligands or charge neutralizing anions which are derived from any monodentate or polydentate ligand or ligand system or its corresponding anion (for example, benzoic acid and benzoate anion, phenolic or phenoxide hp 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, heterocycloalkylamino, heterocycloarylamino, amine oxide, hydrazine, alkylhydrazine, arylhydrazine , 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 acid, alkylthiolcarboxylic acid, arylthiolcarboxylic acid, alkylthiolthiocarboxylic acid, arylthiolthiocarboxylic acid, alkylcarboxylic acid (such as acetic acid, trifluoroacetic acid, oxalic acid), arylcarboxylic acid (such as benzoic acid, phthalic acid), urea, alkylurea, arylurea, alkylarylurea, thiourea , aryithiomochovin, alkylaryltthiomovin, sulphate, sulfur, hydrogens, hydrogensiřičitan, thiosulp, thiosičitan, hydrosičitan, alkylphosfan, arylphosphoshan osfin sulfid, alkylarylfosprinsulfide, alkylphosphorite of acid, aryllphosphorite of acid, alkylphinic, acid, aryl phosphinic acid, alkylphosphinic acid, arylphosphinic acid, phosphate, thiophosphate, phosphorite, pyrophosphite, triphosphate, hydrogen phosphate, dihydrogen phosphate, alkylguanidine, arylguanidine, alkylarylguanidine, alkylcarbamate, arylcarbamate, alkylarylcarbamate, alkylthiocarbamate, alkyldithiocarbamate, aryldithiocarbamate, alkylaryldithiocar bamate, bicarbonate, carbonate, perchlorate, chlorate, chlorite, hypochlorite, bromide, bromate, bromide, bromide, tetrahalomanganate, tetrafluoroborate, hexafluorophosphate, hexafluoroantiammonate, phosphate, iodate, periodate, metaborate, tetraarylborate, tetraalkylborate, tartrate, oxalate, succinate, citrate, ascorbate, saccharinate, amino acid, hydroxamic acid , thioparatoluenesulfonate, and ion exchange resin anions, or systems where one or more X, Y, and Z are independently attached to one or more R" groups, where n is 0 or 1. Preferred ligands for X, Y, and Z include halide, organic acid, nitrate and bicarbonate anions.

The linking groups, here also called linkr", are derived from specified functional groups attached to the groups R or X, Y and Z and functions connecting the biomolecule to the groups "R" or X, Y and Z. The functional groups are selected from the group consisting of -NH ₂ , -NHR ₁₀ , -SH, -OH, -COOH, -COOR ₁₀ , -CONH _2l -NCO, -NCS, COOX”, alkenyl, alkynyl, halide, paratoluenesulfonate, methanesulfonate, tresylate, triflate and phenol , where R ₁₀ is alkyl, aryl, or alkylaryl and "X" is a halide. A commonly preferred alkenyl group is ethenyl and a preferred alkynyl group is ethynyl. The functional groups R or X, Y and Z are reactive with the biomolecule, that is, reactive with the functional group on steroids, bicarbonates, fatty acids, amino acids, peptides, proteins, antibodies, vitramins, lipids, phospholipids, phosphates, phosphites, nucleic acids, enzyme substrates, enzyme inhibitors, enzyme receptor substrates and other target biomolecules. If a functional group is attached to the ”R or X, Y and Z groups, it reacts with the biomolecule, the functional group is modified and this derived functional group is a binding group. For example, if the -NH ₂ functional group attached to the R" group reacts with the steroid of Example 1, the linking group is -NH-. The exact structure of specific binding moieties will be apparent to those skilled in the art and depends on the specific functional group and biomolecule selected. The specific reaction conditions for the reaction of the functional group attached to the "R" or X, Y and Z groups with the biomolecule are obvious to those skilled in the art.

A functional group useful for the formation of a linking group, defined herein as a linking group precursor" may be present on the R" groups when the macrocycle is prepared, or may be added or modified after the macrocycle or manganese complex thereof is prepared. Similarly, the linking group precursor may be present on an axial ligand, that is, on X, Y or Z, if a manganese or iron complex is prepared, or if an exchange reaction of axial ligands is carried out as an exchange of axial ligands present in a manganese or iron complex.

According to the invention, the macrocycle can be formed with manganese or iron before or after conjugation with the target biomolecule, depending on the biomolecule used. A conjugate of a macrocyclic complex and a target biomolecule is defined here as a bioconjugate.

The use of the drugs is generally known to those skilled in the art. See, for example, J. A. Katzenellenbogen et al., Journal of Nuclear Media, Vol. 33, No. 4, 1992, 558, and J.A. Katzenellenbogen et al., Bioconjugate Chemistry, 1991, 2, 353. Targets are commonly biomolecules. Biomolecules according to the invention are biologically active molecules that are locally specific, that is, they are concentrated in a certain organ or tissue. Biomolecules are selected precisely according to the tissue distribution of the bioconjugate through receptor binding, membrane association, membrane solubility, and the like. Biomolecules include, for example, steroids, bicarbonates (including monosaccharides, disaccharides and polysaccharides), fatty acids, amino acids, peptides, proteins, antibodies (including their polyclonal and monoclonal fragments), vitamins, lipids, phospholipids, phosphates, phosphates, nucleic acids, enzyme substrates , enzyme inhibitors and enzyme receptor substrates. Biomolecules also include those biomolecules that are a combination of the biomolecules listed above, for example a combination of a steroid with a bicarbonate, for example digitonin.

Some biomolecules that can be used as targets for a certain organ or tissue are already known or are obvious to those skilled in the art. The biomolecules of the invention are commercially available or can be prepared by a person skilled in the art using conventional methods.

It is usually preferred that at most one Ш" group attached to a carbon atom located between nitrogen atoms in the macrocycle has the macromolecule attached via a linking group. In addition, preferred compounds are those having one to five, preferably one to two, of the R" groups attached to the biomolecule and no X, Y and Z attached to the biomolecule, or those having one X, Y and Z attached to the biomolecule and they do not have an "R" group attached to the macromolecule.

Preferred compounds are usually those where at least one, more preferably at least two of the "R" groups, in addition to the "R" groups attached to the biomolecule, represent alkyl, cycloalkylalkyl and aralkyl radicals and the remaining "R" groups not attached to the molecule represent hydrogen, saturated , a partially saturated or unsaturated ring, or a nitrogen-containing heterocycle. Other preferred groups of compounds are those where at least one, preferably two R 1 or R 1 and R ₂ or R ₂ , R 3 or R ₃ and R 4 or R 4 , R ₅ or R 6 and Re or Re, R ₇ or R? and Re or Re and Re or Rg and R or R together with · ·

9··* * • 9 • · 9

9«

9 99

9·9 9 «

9 9

99 carbon atoms to which they are attached represent a saturated, partially saturated or unsaturated ring with 3 to 20 carbon atoms, and the remaining "R" groups other than R" groups that are attached to the biomolecule by a bonding group that is hydrogen, a nitrogen-containing heterocycle or an alkyl group and those where at least one, preferably two R or R and R 1 or R ₂ or R _{2 1} R ₃ or R 3 and R 4 or R 4 , R ₅ or R 5 and R _e or Re, R ? or R ₇ and R$ or Re and R ₉ or Re together with the carbon atoms to which they are attached are linked to form nitrogen-containing heterocycles with 2 to 20 carbon atoms and the remaining R groups other than the "R" groups attached to the biomolecule by means of a linking group, are independently selected from hydrogen, saturated, partially saturated or unsaturated ring or alkyl groups.

As used herein, ``R'' groups represent all R groups attached to carbon atoms of the macrocycle, that is, R R1, R./., R ₂ , R ₂ ', R3, R3', R4, R<, Rs-, Rs , Re , Rď , R ₇ . R7 3 Re , Re , Ø9, Rg .

Another preferred embodiment of the invention is a pharmaceutical composition in the form of a unit dose, which is useful for superoxide dismutation, containing (a) a therapeutically or prophylactically effective amount of the complex described above and (b) a non-toxic, pharmaceutically acceptable carrier, adjuvant or additives.

The generally accepted mechanism of action of manganese-based SOD enzymes involves transitions of the manganese center between two oxidation states (II, III). See J.V. Bannister. W.H. Bannister and G. Rotilio, Crit. Roar. Biochem., 22, 111-180 (1987).

1) Μη (II) + HO ₂ -> Mn(lll) + HO ₂

2) Mn(lll) + O ₂ -> Μη (II) + O ₂

The formal redox potentials for O ₂ / O ₂ - and HO ₂ / H ₂ O ₂ at pH = 7 are -0.33 V and 0.87 V, respectively. See AEG Cass, in Metalloproteins; Part 1, Metal proteins with Redox Roles, ed. P. Harrison, P. 121. Verlag Chemie (Weinheim, GDR) (1985). In the mechanism above, these potentials require the putative catalyst to be able to rapidly transition through a change in oxidation state ranging from -0.33V to 0.87V.

The complexes described here derived from Μη (II) and in general the class of C-substituted ligands [15]anN _s were characterized using cyclic voltammetry by measuring their redox potential. The C-substituted manganese-based complexes described here have a reversible oxidation of approximately +0.7V (SHE). Coulometry shows that this oxidation is a ♦ · · • ·*·· ·*· one-electron process; more precisely, it is the oxidation of the manganese complex to the manganite complex. For these complexes, for their function as SOD catalysts, the oxidation state Μη (III) is included in the catalytic cycle. This means that the maganite complexes of all * * these ligands are equally competent as SOD catalysts, regardless of which form (Μη (II) or Mn(lll)) is present in the presence of superoxide, since superoxide reduces Μη (III) to Μη (II) for the release of oxygen.

The iron-based complexes of the invention are useful in part due to the unexpectedly increased stability of the iron-based complexes compared to the corresponding manganese-based complexes. This increased stability may be important for oral administration and where the target tissue has a very low pH, such as cardiac tissue.

As used herein, the term "alkyl" alone or in combination, means a straight or branched chain alkyl radical containing U. up to 22 carbon atoms preferably 1 to about 18 carbon atoms and most preferably 1 to about 12 carbon atoms optionally bearing one or more substituents selected from (1) -NR30R31 wherein R30 and R31 are independently selected from hydrogen, alkyl, aryl or aralkyiu; or R ₃₀ is hydrogen, alkyl, aryl or aralkyl and R 31 is selected from the group consisting of

-NR32 R33, -OH, -OR34,

*5

X

II

-P-CO^jXORjj);

where R 32 and R 3 are independently hydrogen, alkyl, aryl or acyl, R 3 is alkyl, ary! or alkaryl, Z' is hydrogen, alkyl, aryl, alkaryl, - R34, -SR34 or -NR40 R41, where R40 and R41 are independently selected from hydrogen, alkyl, aryl or alkaryl, Z" is alkyl, aryl or alkaryl, - OR34, SR34, -NR40 R41, -R35, is alkyl, aryl,

-OR>,, -NR 40 R 41 , R 36 is alkyl, aryl or -NR 40 R 41 . R 37 is alkyl, aryl or alkaryl, X' is oxygen or sulfur and R 3 and R ₃₉ are independently selected from hydrogen, alkyl, aryl; (2) -SR 42 where R 42 is hydrogen, alkyl, aryl or alkaryl, -SR 34 , -NR ₃₂ R 33 ,

X o o — Ϊ:-Ζ\ -S-^43, OT -?4AXB);

·4· ··· · · • ·*·« * «4 • ··♦· 4

4

4· 44 where R 34 is -OH, -OR 34 or -NR ₃₂ R», and A and B are independently -OR 34 , -SR 34 or -NR 32

R33;

(3) wherein x is 1 or 2, and R 44 is halide, alkyl, aryl or alkaryl, -OH, -OR 34 , -SR 34 , -NR _K R 33 ;

(4) -OR 45 where R 45 is hydrogen, alkyl, aryl, alkaryl, -NR ₃₂ R 33 ;

X' 0 0 II — C—Z', -8t=flki -P-PXE),or

wherein D and E are independently -OR 34 or -NR ₃₂ R 33 ;

wherein R 46 is halide, -OH, -SH, -OR 3 , -SR 34 or -NR ₃₂ R 33 ; or (6) an amine oxide of the formula —N R30R3:

with the proviso that R 30 and R 3 are not hydrogen; or (7) wherein F and G are independently -OH, -SH, -OR 34 , -SR 3 , -NR 32 R 33 ; or

4« *4 * · · · * · ·· · • 4 4

4 • 4 4 4

4··

444 4 4 . 4 4 4

44 (8) -O-(-(CH ₂ ) _a -O) _b -Rw, wherein R ₁₀ is hydrogen or alkyl, aa and b are integers independently selected from the group of 1 to 6; or (9) halogen, cyano, nitro or azido. Alkyl, aryl and alkaryl groups substituted for the alkyl groups mentioned above may contain one additional substituent, but are preferably unsubstituted. Examples of such radicals include, but are not limited to, 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, represents an alkyl radical having one or two double bonds. Examples of such alkenyl radicals include, but are not limited to, ethenyl, propenyl, 1-butenyl, cis-2-butenyl, trans-2-butenyl, iso-butenyl, cis-2-pentenyl, trans-2-pentenyl, 3-methyl- 1-butenyl, 2,3-dimethyl-2-butenyl, ipentenyl, 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” • A represents, alone or in combination, an alkyl radical with one or more triple bonds. Examples of such alkynyl moieties include, but are not limited to, ethynyl, propynyl (propargyl), 1-butynyl, 1-octynyl, 9-octadecinyl, 1,3-pentadiinyl, 2,4pentadiinyl, 1,3-hexadiinyl, and 2,4- hexadiinesF. The term "cycloalkyl" represents, alone or in combination, a cycloalkyl radical containing 3 to approximately 10, preferably 3 to 8 and most preferably 3 to 6 carbon atoms. Examples of such cycloalkyl radicals include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and perhydronaphthyl. The term "cycloalkylalkyl" represents an alkyl radical as defined above which is substituted with a cycloalkyl radical as defined above. Examples of cycloalkylalkyl radicals include, but are not limited to, cyclohexylmethyl, cyclopentylmethyl, (4-isopropylcyclohexyl)methyl, (4-t-butyl-cyclohexyl)methyl, 3-cyclohexylpropyl, 2-cyclohexylmethylpentyl, 3-cyclopentylmethylhexyl, 1-(4-neopentylcyclohexylmethylhexyl), and 1 -(4-isopropylcyclohexyl)" means a cycloalkyl radical as defined above. Examples of cycloalkylcycloalkyl radicals include, but are not limited to, cyclohexylcyclohexyl, alone or in combination a cycloalkyl radical with one or more double bonds Examples of cycloalkenyl radicals include, but are not limited to, cyclopentenyl, cyclohexenyl, cyclooctenyl12 « · • · · • ««·* * • · ·· · »1 * «·· · * • ♦ · ·· ·· nyl, cyclohexadienyl and cyclooctadienyl The term cycloalkenylalkyl represents an alkyl radical as defined above which is substituted with a cycloalkenyl radical as defined above. Examples of cycloalkenylalkyl radicals include, but are not limited to, 2-cyclohexen-1-ylmethyl, 1-cyclopenten-1-ylmethyl, 2-(1-cyclohexen-1-yl)ethyl, 3-(1-cyclopenten-1-yl)propyl, 1- {1-cyclohexen-1-ylmethyl)pentyl, 1-(1-cyclopenten-1-yl)hexyl, 6-(1-cyclohexen-1-yl)hexyl, 1-(1-cyclopenten-1-yl)nonyl and 1- (1-cyclohexen-1ylnonyl and 1-(1-cyclohexen-1-yl)nonyl. The terms "alkylcycloalkyl" and "alkenylcycloalkyl" mean a cycloalkyl radical as defined above which is substituted with an alkyl or alkenyl radical as defined above. Examples of alkylcycloalkyl and alkenylcycloalkyl radicals include, but are not limited to

2-ethylcyclobutyl, 1-methylcyclopentyl, 1-hexylcyclopentyl, 1-methylcyclohexyl, 1-(9-octadecenyl)cyclopentyl.. and .1-(9-octadecenyl)cyclohexyl. The terms "alkylcycloalkenyl" and alkenylcycloalkenyl" mean a cycloalkenyl radical as defined above which is substituted with an alkyl or alkenyl radical as defined above. Examples of alkylcycloalkenyl and alkenylcycloalkenyl radicals include, but are not limited to, 1-methyl-2-cyclopentenyl, 1-hexyl-2-cyclopentenyl, 1-ethyl-2-cyclohexenyl _; 1-butyl 2-cyclohexenyl, 1-(9-octadecenyl)-2-cyclohexenyl and 1-(2-pentenyl)-2-cyclohexenyl. The term aryl" alone or in combination, represents a phenyl or naphthyl radical, which is optionally carried by one or more substituents selected from the group of alkyl, cycloalkyl, cycloalkenyl, aryl, heterocycle, alkoxyaryl, alkaryl, alkoxy, halogen, hydroxyl, amine, cyano, nitro , alkylthio, phenoxy, ether, trifluoromethyl and the like, for example phenyl, ptolyl, 4-methoxyphenyl, 4-(tert-butoxy)phenyl, 4-fluorophenyl, 4-chlorophenyl, 4-hydroxyphenyl, 1-naphthyl, 2-naphthyl, and the like. The term "aralkyl" alone or in combination, represents an alkyl or cycloalkyl radical as defined above wherein one hydrogen atom is replaced by an aryl radical as defined above, such as benzyl, 2-phenylethyl, and the like. The term "heterocyclic" means a cyclic structure containing at least one other type of atom besides carbon in the ring. The most commonly represented other type of atom includes nitrogen, oxygen, and sulfur. Examples of heterocycles include, but are not limited to, pyrrolidinyl, piperidyl, imidazolidinyl, tetrahydrofuryl, tetrahydrothienyl, furyl, thienyl, pyridyl, quinolyl, isoquinolyl, pyridazinyl, pyrazinyl, indolyl, imidazolyl, oxazolyl, thiazolyl, pyrazolyl, pyridinyl, benzoxadiazolyl, benzothiadiazolyl, triazolyl, and tetrazolyl groups. The term saturated, partially saturated or unsaturated cycle represents connected ring structures where two carbon atoms of the ring are also part of * * · • · ···· * • · · ·

«9 9

9, 99 ··♦ · 9 • 9 ♦

99' 99 fifteen-membered macrocyclic ligand The ring structure may also contain 3 to 20 carbon atoms, preferably 5 to 10 carbon atoms, and may also contain one or more other types of atoms in addition to carbon. The most commonly represented other type of atom includes nitrogen, oxygen, or sulfur. A ring structure can also contain more than one ring. The term "saturated, partially saturated or unsaturated cyclic structure" represents a ring structure where one carbon of the ring is also part of a fifteen-membered tmacrocyclic ligand. The ring structure may contain 3 to 20 carbon atoms, preferably 5 to 10 carbon atoms, and may also contain nitrogen, oxygen and/or sulfur atoms. The term nitrogen-containing heterocycle means a ring structure where two carbon and nitrogen atoms of the ring are also part of a fifteen-membered macrocyclic ligand. The ring structure can also contain 2 to 20 carbon atoms, preferably 4 to. 10 carbon atoms, can be partially or completely unsaturated or saturated and can also contain nitrogen, oxygen and/or sulfur atoms in that part of the ring that is not also part of the fifteen-membered macrocyclic ligand. The term organic acid anion means carboxylic acid anions with 1 to 18 carbon atoms. The term "halohydride" means chloride or bromide.

The macrocyclic ligands useful in the complexes of the invention can be prepared according to the general procedure shown in Scheme A below. An amino acid amide that corresponds to an amide derivative of a naturally or unnaturally occurring α-amino acid is reduced to the corresponding substituted ethylenediamine form. This amino acid amide may be an amide derivative of any of a number of generally known amino acids. Preferred amino acid amides are represented by the formula:

where R is a derivative of 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 R groups of unnatural α-amino acids, such as alkyl, ethyl, butyl, tert-butyl, cycloalkyl, phenyl, alkenyl, allyl, alkynyl, aryl, heteroaryl, polycycloalkyl, polycycloaryl, polycycloheteroaryl, imine, aminoalkyl, hydroxyalkyl,

w» * w V « t t · » · · ··!*·· * · ·*·· · · · · ·*· · · ··· · * ··· • I · «···· ·· · hydroxyl, phenol, amine oxide, thiolalkyl, carboalkoxyalkyl, carboxylic acids and their derivatives, keto, ether, aldehyde, amine, nitrile, halogen, thiol, sulfoxide, sulfone, sulfonic acid, sulfide, disulfide, phosphonic acid, phosphinic acid, phosphine oxide, sulfonamide, amide, amino acid, peptide, protein, bicarbonate, nucleic acid, fatty acid, lipid, nitro, hydroxylamine, hydroxamic acid, thiocarbonyl, borate, borane, borase, silyl, siloxy/silase, and combinations thereof. Those where R represents hydrogen, alkyl, cycloalkanyl and aralkyl radicals are best. The diamine is then tosylated to form a di-N-p-toluenesulfone derivative, which reacts with a di-O-tosylated tris-Ntosylated triazalakane diol to form the corresponding substituted Npentatosylpentazacycloalkane. The tosylating groups are then removed and the resulting compound is reacted with a manganate or ferric compound under the necessary anhydrous and anaerobic conditions to form the corresponding substituted manganate or ferric pentaazacycloalkane complex. If the ligands or charge are neutral with lysing anions, that is, X, Y, and Z anions or ligands that cannot come directly from the manganese or iron compound, the complex of these anions or ligands can be formed by performing an exchange reaction with the complex that was prepared by the reaction macrocycle with a manganese or iron compound.

Complexes according to the invention, where Rs and R ₂ are alkyl and R ₃ , R'3, R4, R/Rs, R'5, Re, R'e, R7, R7, Rs and Rs can be alkyl, arylalkyl or cycloalkylalkyl and R or R' and Rí or R'i are bonded together with the carbon atoms to form a nitrogen-containing heterocycle may also be prepared according to the general procedure outlined in Scheme B below using methods known to those skilled in the art, for the preparation of the manganese precursor nabo of the ferric pentaazadicyclo[12,3,1]octadecapentane complex. See, for example, Alexander et al., Inorg. Nucl. Chem. Lett., 6, 445 (1970). Thus, 2,6-diketopyridine condensed with triethylenetetramine in the presence of a manganese or iron compound to form a manganese or iron peritaazadicyclo[12,3,1]octadecapentane complex. The manganese(II) iron pentaazadicyclo[12,3,1]octadecapentane complex was hydrogenated with platinum oxide at a pressure of 68.8 kPa -6888.8 kPa to give the corresponding manganese(II) iron pentaazadicyclo[12,3,1]octadecapentane complex or metal (lil).

The macrocyclic ligands useful in the complexes according to the invention can also be prepared by the dichloride route shown in Scheme C below. The triazalkane is thus tosylated in a suitable solvent system to form the corresponding tris(N15 *·· ·· 44 4»·*

4 4 4 4 4 4 ·>.. 44 • · 4 4444 · * 4 · ·· 4 4 ··· 4 4 4 4 4 «4 « 444 444 44 *4 p-toluenesulfone) deriv. This derivative reacts with an appropriate base to form the corresponding disulfonamide anion. The disulfonamide anion is dialkylated with a suitable electrophile to form a dicarboxylic acid derivative. This dicarboxylic acid derivative reacts to form a dicarboxylic acid, which then reacts with a suitable reagent to form a dichloride. The desired vicinal diamine is obtained by one of the possible routes. One route is useful for preparation from aldehyde by reaction with cyanide in the presence of ammonium chloride and subsequent reaction with acid to form alpha ammonium nitrile. The latter compound is reduced in the presence of an acid and then reacted with a suitable base to form a vicinal diamine. Condensation of the dichloride of a dihydric acid with a vicinal diamine in the presence of a suitable base gives rise to the tris(p-toluenesulfone)diamine macrocycle. The p-toluenesulfone group is removed, the amides are reduced, and the resulting compound reacts with manganate, or a ferric compound under the necessary anhydrous and anaerobic conditions and the formation of the corresponding substituted manganese or ferric pentaazacycloalkane complex.

Vicinal diamines are prepared by the indicated route (known as the Strecker synthesis) and vicinal diamines are purchased when commercially available. Any method can be used to prepare the vicinal diamine.

The macrocyclic ligands useful in the complexes of the invention can also be prepared by the pyridinediamide route shown in the following Scheme D below. The polyamine, as a tetraaza compound containing two primary amines, thus condenses with dimethyl-(2,6-pyridinedicarboxylate) on heating in a suitable solvent, such as methanol, to form a macrocycle with an incorporated piperidine ring as a 2,6-dicarboxamide. The pyridine ring in the macrocycle is reduced to the corresponding piperidine ring in the macrocycle, the diamines are then reduced, and the resulting compound reacts with a manganate or ferric compound under the necessary anhydrous and anaerobic conditions to form the corresponding substituted manganate or ferric pentaazacycloalkane complex.

The macrocyclic ligands useful in the complexes according to the invention can also be prepared by the bis(haloacetamide) route shown in Scheme E below. The triazaalkane is thus tosylated in a suitable solvent system to form the corresponding tris(N-ptoluenesulfone) derivative. This derivative then reacts with a suitable base to form the disulfonamide anion. A bis(haloacetamide), such as bis(chloroacetamide) of vicinal diamide, is prepared by reacting a diamine with an excess of haloacetyl halide, for example with ·· • · • 4 ♦ * 4444 • 4 · « · » • 4 · ·· « 44* 4 4

4 ♦ · · • 4« 444 * «· ·· chloroacetyl chloride, in the presence of a base. The disulfonamide anion of tris(N-ptoluenesulfone)triazaalkane then reacts with bis(chloroacetamide)diamine to form a substituted tris(N-tosyl)diamide macrocycle. The p-toluenesulfone groups are removed, the amides are reduced, and the resulting compound is reacted with a manganate or ferric compound under the necessary anhydrous and anaerobic conditions to form the corresponding substituted manganate or ferric pentaazacycloalkane complex.

Macrocyclic ligands usable in the complexes according to the invention, where R _b R,', R ₂₎ R ₂ are derived from a diamine starting substance and R ₅ , R ₅ ', R ₇ , R? and R ₉ , R ₉ ' can be H or any functional group mentioned above can be prepared by the pseudopeptide method shown in Scheme F below. Substituted 1,2-diaminoethane represented by the formula ........ .........

where R 1 , R y , R ₂ and R ₂ are substituents on adjacent carbon atoms of the macrocyclic ligand product as above, and may be used in this method in combination with any amino acid. The diamine may be prepared by any commonly used method known to those skilled in the art. The R groups in the macrocycle, derived from the substituents on the α-carbon of α-amino acids, that is, Rs, Rs', R ₇ , R ₇ ', Rg and Rg', may be derived from the D or L forms of the amino acid alanine, aspartic acid, arginine ^asparagine, cysteine, glycine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, proline, phenylalanine, serine, tryptophan, threonine, tyrosine, valine and/or R groups of unnatural α-amino acids such as alkyl, ethyl, butyl, tert.butyl, cycloalkyl, phenyl, alkenyl, allyl, alkynyl, aryl, heteroaryl, polycycloalkyl, polycycloaryl, polycycloheteroaryl, imine, aminoalkyl, hydroxyalkyl, hydroxyl, phenol, amine oxide, thiolalkyl, carboalkoxyalkyl, carboxylic acids and their derivatives, keto, ether, aldehyde, amine, nitrile, halogen, thiol, sulfoxide, sulfone, sulfonic acid, sulfide, disulfide, phosphonic acid, phosphinic acid, phosphine oxide, amide, amino acid, peptide, protein, bicarbonate, nucleic acid, fatty acid, lipid, nitro , hydroxylamine, hydroxamic acid, thiocarbonyl, borate, •

borane, borase, silyl, siloxy, silase, and combinations thereof. For example, 1,8-dihydroxy, 4,5diaminooctane is monotosylated, and reacts with Boc anhydride to form different NBoc, Np-toluenesulfone derivatives. The sulfonamide is alkylated with methyl bromoacetate using sodium hydride as a base and saponified to the free acid. The diamine containing N-ptoluenesulfonyglycine is used as a replacement for the dipeptide in standard solution-based peptide synthesis. By combining with an amino acid ester bearing a functional group, the corresponding pseudo-tripeptide is formed. Two consecutive reactions, TFA cleavage-coupling, yield a pseudopentapeptide from which the terminal N- and C- groups can be removed in one step using HCl/AcOH. DPPA-mediated cyclization with subsequent reduction of LiAIH ₄ or boron yields the corresponding macrocyclic ligand. This ligand system reacts with a manganate or ferric compound, such as manganese chloride or ferric chloride, under the necessary anhydrous and anaerobic conditions to form the corresponding manganate or ferric pentaazabicycloalkane complex. If the ligands or charge neutralizing anions, that is, X, Y, and Z are anions or ligands that cannot come directly from a manganese or iron compound, a complex with these anions or ligands can still form by performing an exchange reaction with a complex that was prepared by reacting the macrocycle with a manganese or iron compound.

The macrocyclic ligands useful in the complexes of the invention, where R _1f R 1 , R 3 , R 3 , R s , R s , R 7 , R 1 , R s and R 8 , can be H or any functional group mentioned above, and can be prepared according to the general peptide method shown in the scheme G below. The R groups in the macrocycle derived from the substituent on the α-carbon of the α-amino acids, that is Rí, Rí, R3.R3.R5, Rí, R7, R7', R7', R7 and R9 are defined above in Scheme F. The procedure for the preparation of cyclic peptide precursors from the corresponding of linear peptides are the same, or with an obvious variation of already known methods. See, for example, Veber, DF et al., J. Org. Chem., 44. 3101 (1979). The general procedure of the method in Scheme G below exemplifies the use of a solution-based preparation of a functionalized linear N-terminal to C-terminal pentapeptide. The reaction procedure for the preparation of the linear pentapeptide can optionally be carried out on a solid phase basis using already known methods. The reaction can proceed from the C-terminus to the N-terminus, and the conversion can achieve fusion of di- and tri-peptides, if necessary. The Boc-protected amino acid is thus coupled to the amino acid ester using standard peptide coupling reaction reagents. The new Boc-dipeptide ester is then saponified into a free acid, which combines again with another amino acid ester18 • · ♦ * ···· * lin. The resulting Boc-tri-peptide ester is again saponified and this method continues until the preparation of the Boc-protected pentapeptide free acid. The Boc-protecting group is removed under standard conditions and the resulting pentapeptide or its salt is converted to a cyclic pentapeptide. The cyclic pentapeptide is then reduced to pentaazacyclopentadecane with lithium aluminum hydride or boron. The last ligand then reacts with a manganate or iron compound under the necessary anaerobic conditions to form the corresponding manganate or iron pentaazacyclopentadecane complex. If the ligands or charge neutralizing anions, that is, X, Y, and Z are anions or ligands that cannot directly originate from a manganese or iron compound, a complex with these anions or ligands can be formed by performing an exchange reaction with a complex that was prepared by reacting the macrocycle with a manganese or iron compound.

The following schemes illustrate the preparation of manganese complexes according to the invention. The iron complexes according to the invention can be prepared by replacing the manganese compound used with an iron compound.

* «

SCHEME A

0 ·0 • * · • 0000 ·

0 00 00 0 0

• ·

999 9 9

9

No^-CHO

SCHEME C

I.TtCI, .

py*fin _Ts ^N

1. KCN, NH ₄ a

NH.OH.tttf

2. HCT

BřCRjCOOCHj dtrf

Γγ-^· R' _e « H HsN+ci“ j Hy PtOý-Ha ...... R 6 R r ₉ 4_P'' * ^r - ^h

CI'HsN+ Aftťcr base °yOCH ₃ CH,oU ⁵

8-J L-*

HaN NHj

CICOCOCI 0

o.srW/H

or thf

AND

H

• 4

SCHEME D • · · • »··♦ 4 •

• 4 4 • 44 • 4 4 · • •4 · « • · 4 · ·4

H ₂ .PtO _ø CHgOH HO

000 • * · • 0*0« 0 • Β0·« · 0000 « 0 ·00 •0 0« 0«

SCHEME E

»H V.

·»· » ^r · ' • · 9 · « « ϊ

♦ ··

·3

SCHEME F

F •V'??'

4 4 44 *· 4 4»» • 4 · · · · 4 4 · · · 444· · · · · ·· 4 4

4« 4 · 4 « I • 4 · 44444« 44» ··

SCHEME F {continued)

• 0

SCHEME G * 0 0 « 0 0 0 • 0 000 •00 ·« · ·· «000 * · 000« • 0 0 00 0 · · • 0 0 0 0 •0 000 00 ··

QOOVf

NaOKHaQ

CHjOH

V· I i

OQCNn

R i

BocNH

El edc-hci, HOBT, OMF.TCA,RT or +

NaOH, HjO CHjOH

BocNH

Ethyl cMoroformate, ' dmf,tea,o*c

'Et

O

EDOHCI, HOBT, dmf.tea.rt or

Ethyl cWwofonnat , DMF,TEA.0®C • φ • · φ φ φ · φφφφ φ • φ φ φφ φ φ

φφφ · • φφ φφ φ φ φ • φ φ φφ φφ

SCHEME G (continued)

ΜΟ^, ΜβΟΗ

* · 9 ^o'clock ··..

4' 9 99 99 9 • *£? · · ·

099 999

The pentaazamacrocycles according to the invention may have one or more asymmetric] carbon atoms and are thus capable of existing in the form of optical isomers, the same as the form of their racemic or non-racemic mixtures. Optical isomers can be obtained ⁷ ·

T from racemic mixtures by conventional methods, for example by the formation of diastereomeric salts by reaction with an optically active acid. Examples of suitable acids are tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, ditoluenetartaric acid and camphorsulfonic acid. Then follows the separation of the mixture of diastereoisomers by crystallization with subsequent separation of the optically active sludge from these salts. Various procedures for the separation of optical isomers involve the use of a chiral chromatographic column optimally selected for maximum separation of enantiomers. Another applicable method involves the synthesis of covalent diastereomeric molecules by reacting one or more secondary amino group(s) of the compounds according to the invention with an optically pure acid in an activated form or an optically pure isocyanate. The synthesized diastereomers can be separated by conventional methods, such as chromatography, distillation, crystallization, or sublimation, and then hydrolyzed to a separate enantiomerically pure ligand. Optically active compounds according to the invention can also be obtained from optically active starting materials, such as for example from natural amino acids.

The compounds or complexes of the invention are novel and may be used in the treatment of many inflammatory disease states and disorders. For example, ischemic organ reperfusion injury such as myocardial ischemic reperfusion injury, surgical ischemia, inflammatory bowel disease, rheumatoid arthritis, osteoarthritis, psoriasis, organ transplant rejection, radiation-induced injury, oxidation-induced tissue injury and damage, atherosclerosis, thrombosis, aggregation platelets, stroke, acute pancreatitis, insulin-dependent diabetes mellitus, scattered intravascular coagulation, fat embolism, respiratory failure in adults and children, metastasis and carcinogenesis.

The activity of compounds or complexes of the invention in catalyzing superoxide dismutation can be explained using the technique of stopped-flow kinetic analysis as described in Riley, DP, Rivers, WJ and Weiss, RH, Stopped-Flow Kinetic Analysis for Monitoring Superoxide Decay in Aqueous Systems, Anal. Biochem.. 196, 344-349 (1991), which is incorporated herein by reference. Stopped flow kinetic analysis is an accurate and direct method for quantitatively monitoring the rate of decomposition of superoxide in water. Cinemas' *τ* ^: · · ♦ ι«β t ta I V 1

->n ·Η·«··· · » · * ····*' ·>··|Μ· ' · * ixl«Pl '-'··· *«FÍ^· · ··· '· · *·^··I tical stopped-flow analysis is suitable for screening compounds with SOD activity by the catalytic activity of compounds or complexes according to the invention for dismutation of superoxide, as shown by stopped-flow analysis, correlated with the treatment of the above-mentioned disease states and disorders. The total daily dose administered to the patient in a single dose or in several doses can range, for example, from 1 to about 100 mg/kg of body weight per day, and usually from about 3 to 30 mg/kg. The composition of the unit dose should contain an amount several times lower than the daily dose.

The amount of active substance that can be mixed with the carrier to obtain a unit dose can vary depending on the patient being treated and the method of administration.

Dosage for the treatment of disease states with the compounds and/or compositions of the invention depends on many factors, including the type, age, weight, sex, aging regime and medical condition of the patient, degree of disease; the frequency of administration, pharmacological aspects such as the activity, efficacy, pharmacokinetic and toxicological profile of a particular compound, the drug used in the administration system, and whether the administered compound is part of a drug combination. There are so many differences in the actual dosage system and it may therefore differ from the preferred dosage system mentioned above.

The compounds according to the invention can be administered orally, parenterally, by inhalation spray, rectally, or locally in the form of a unit dose containing common, »

non-toxic, pharmaceutically acceptable carriers, possibly adjuvants and additives. Topical administration may also include transdermal application, for example transdermal patches or iontophoretic agents. The term parenteral as used herein includes subcutaneous injection, intravenous, intramuscular, intrasternal injection, or infusion techniques.

Injectable preparations, such as sterile water for injection or oil suspensions, can be prepared according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example a solution in 1,3-butanediol. Acceptable carriers and solvents that may be used include water, Ringers solution, and isotonic. sodium chloride solution. In addition, sterile, fixed oils are commonly available as solvents or suspending media. Any mixture of fixed oil including synthetic mono- or diglycerides can be used for this purpose. Fatty acids, for example oleic acid, have also found application in the preparation of injectable substances.

3o :

444 4 4 44««

44«« 4 · * · ··« 4 · « « « · « 4 *

4 »4« «·4 4« 4«

Suppositories for rectal administration of the drug can be prepared by mixing the drug with a suitable non-irritating carrier, such as cocoa butter and polyethylene glycol, which are solid at room temperature but liquid at rectal temperature and therefore melt and release the drug in the rectum.

Solid dosage forms for oral administration may include capsules, tablets, pills, powders, granules, and gels. In these solid forms, the active compound may be mixed with at least one diluent, for example lactose sugar or a resin. Such dosage forms may also commonly contain other substances in addition to inert diluents, for example lubricating agents, for example magnesium stearate. In the case of capsules, tablets and pills, dosage forms may also contain buffering substances. Prepared tablets and pills may additionally have an enteric coating.

Liquid forms for oral administration- may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and tinctures containing commonly used inert solvents such as water. Such compositions may also contain additives, for example humectants, emulsifying and suspending agents, and sweetening, coloring and flavoring agents.

While the compounds of the invention may be administered as a single active pharmaceutical agent, they may also be used in combination with one or more compounds known to be active agents in a particular disease state, one of which has a therapeutic effect.

The compounds or complexes according to the invention can also be used as MRI contrast agents. A discussion of the use of contrast agents in MRI can be found in patent application number 08/397,469, which is incorporated herein by reference.

Presumed equivalents of the common forms given above for compounds and derivatives, as well as for intermediates, are compounds otherwise corresponding thereto, having the same general properties as tautomers of the compounds and those where one or more of the different R groups are simple permutations of substituents as defined herein, for example, where R is a higher alkyl group than indicated, or where the para-toluenesulfone groups are other nitrogen or oxygen protecting groups, or where the o-para-toluenesulfone is a halide. Anions with a charge other than 1, i.e. carbonates, phosphates and hydrogen phosphates, can be used in place of anions with a charge of 1 if they do not otherwise affect the overall activity of the complex. However, the use of anions with a charge other than 1 causes a slight modification of the general formula of the complex indicated

Φ » « I * · · »· · » • b · • * Φ • Φ· · » * above. Additionally, if the substituent is designed to be, or may be, hydrogen, the exact chemical nature of the non-hydrogen substituent at that position, such as a hydrocarbon residue or a halide, hydroxy, amino, and similar functional group, is not critical as long as it does not otherwise affect the general activity and/ or synthesis procedure. Manganite and iron complexes are further assumed to be equivalent to manganese and iron complexes.

The chemical reactions described above are generally presented in terms of the application of their wider activity for the preparation of compounds according to the invention. In some cases, reactions are not applicable as described for every compound listed. Those skilled in the art will recognize these compounds. In all such cases, the reaction is carried out with the usual modifications known to those skilled in the art, for example by suitable protection of interfering groups, conversion to alternative; by common substance/routine modification of reaction conditions and the like, or by other reactions mentioned herein, or by any other common method applicable to the preparation of the corresponding compounds according to the invention. All preparation methods, all starting materials are known, or they can be easily prepared from starting materials.

Without further description, it is believed that one skilled in the art can utilize the present invention to its fullest extent using the foregoing description. The following advantageous designs are therefore only illustrative and are not restrictive in any way.

Examples of embodiments of the invention

All reagents were used as they were without purification unless otherwise stated. All NMR spectra were obtained on a Varian VXR-300 or VXR-400 nuclear magnetic resonance spectrometer. Qualitative and quantitative mass spectroscopy was performed on Finigan MAT90 and Finigan 4500 and VG40250T using m-nitrobenzyl alcohol (NBA) and m-nitrobenzyl alcohol/LiCl (NBAli) Melting points (mp) unadjusted.

The following abbreviations relating to amino acids and their protecting groups are in accordance with the IUPAC-IUB Commission on Biochemical Nomenclature (Biochemistry 1972,11,1726) and general usage.

Ala L-Alanine

She gave D-alanine

Gly glycine

ΦΦΦ * Μ»

Ser

DSer

Bzl

Bye

Et

TFA

DMF

HOBT. H ₂ O

EDC. HCl

TEA

DMSO

THF

DPPA

L-serine

D-serine benzyl tert -butoxycarbonyl ethyl trifluoroacetic acid dimethylformamide

1-Hydroxy-((H)-benzotriazole monohydrate

-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride triethylamine dimethylsulfoxide tetrahydrofuran - - -.......

diphenylphosphoryl azide 'The abbreviation Cyc stands for 1,2-cyclohexanediamine (stereochemistry, that is, the use of R,R or S,S). The following is the three-letter code of the peptide nomenclature used for pseudopeptides containing a 1,2-cyclohexane diamine residue”.

Example 1

A, Synthesis of N-(p-toluenesulfonyl)-(R.R)-1,2-diaminocyclohexane

To a stirred solution of (R,R)-1,2-diaminocyclohexane {300 g, 2.63 mmol) in

CH ₂ Cl ₂ (5.00 L) was added dropwise at -10 °C over 7 h, maintaining the temperature at -5 °C to -10 °C, a solution of p-toluenesulfonyl chloride (209 g, 1.10 mol) in CH ₂ Cl ₂ (5.00 L) The mixture was warmed with stirring overnight. to room temperature. The mixture was concentrated in vacuo to a volume of 3 L and the white solid was removed by filtration. The solution was then washed with H ₂ O (10x1 L) and dried over MgSO ₄ . Removal of the solvent in vacuo afforded 286 g (97.5% yield) of product as a yellow crystalline solid: ¹ H NMR (CDCfej δ 0.98-1.27 (m, 4H), 1.564-1.66 (m, 2H), 1.81-1.93 (m, 2H), 2.34 (dt, J = 4.0, 10.87 Hz, 1H), 2.42 (s, 3H), 2.62 (dt , J = 4.2, 9.9 Hz, 1H), 7.29 (d, JH = 8.1 Hz, 2JH), 7.77 (d, J = 8.3 Hz, 2H); MS (LRFAB -DTT-DTE) m/z 269 [M + Hf.

B, Synthesis of N-(p-toluenesulfonvO-N'-(Boc)-(R,R)-1,2-diaminocyclohexane

9 9 «

• . *

To a stirred solution of N-(p-toluenesulfonyl)-(R,R)-1,2-diaminocyclohexane prepared in Example 1A (256 g, 0.955 mol) in THF (1.15 L) was added a 1N solution of aqueous NaOH (1.15 L, 1.15 mol). Di-t-butyl dicarbonate (299 g, 1.05 mol) was added and the resulting mixture was stirred overnight. The layers were separated and the aqueous layer was adjusted to pH 2 with 1N HCl and saturated with NaCl. The aqueous solution was extracted with CH ₂ Cl ₂ (2 x 500 mL), the extracts and the THF layer were combined and dried over MgSO ₄ . The solvent was removed in vacuo to give a yellow solid. The crude product was purified by crystallization from a mixture of THF-ether-hexane and 310 g (88.1% yield) of the product was obtained in the form of a white crystalline solid: melting point: 137°C - 139°C : ¹ H NMR (CDCl ₃ ) δ 1.04-1.28 (m, 4H), 1.44 (s, 9H), 1.61-1.69 (m, 2H), 1.94-2.01 (m, 2H), 2 .43 (s, 3H), 2.86 (broad s, 1H), 3.30 (broad d, J = 9.6 1H), 4.37 (broad d, J = 6.7 Hz, 1H), 5.48 (wide d, J = 4.6 Hz, 1H), J.27 (d,. 1=..9.7-Hz, 2HX 7.-73 (d, J=-8.1Hz;- 2H);-MS(LRFAB,NBA ^- -Li)W· 375 [M+· Li]*.

C. Synthesis of Boc-(R,R)-Cyc(Ts)-Gly-OMe

To a stirred mixture of N-(p-toluenesulfonyl)-N'-(Boc)-(R,R)-1,2diaminocyclohexane prepared in Example 1B (310 g, 0.841 mol) in anhydrous DMF (3.11 L) at 0 °C, NaH (37.4 g-60% in oil, 0.934 mol) was added portionwise and the resulting mixture was stirred for 30 min. Methyl(bromoacetate) (142 g, 0.925 mol) was added dropwise over 45 min and the mixture was warmed to room temperature with stirring overnight. After stirring for 17 h, the solvent was removed in vacuo and the residue was dissolved in ethyl acetate (3 L) and H ₂ O (1 L). The ethyl acetate solution was washed with saturated NaHCO ₃ (1 L), saturated NaCl (500 mL) and dried over MgSO ₄ . The solvent was removed in vacuo and the remaining oil was dissolved in ether. Crystallization after addition of hexane afforded 364 g (98% yield) of the product (TLC (98:2 CHCl ₃ -MeOH/silica gel (UV detn) showed that the product contained 5% starting material) in the form of colorless needles: m.p. of the pure sample 151°C-152°C; ^1H NMR (CDFe) δ 1.11-1.22 (m, 4H), 1.45 (s, 9H), 1.64-1.70 (m, 3H), 2.16-2.19 (m, 1H), 2.43 (s, 3H), 3.34-3.40 (m, 2H), 3.68 (s 3H), 4.06 (ABq, J = 18.5 Hz, Δν = 155 Hz, 2H), 4.77 (broad s, 1000H), 7.30 (d, J = 8.3 Hz, 2H), 7.82 (d, J = 8, 3 Hz, 2H); MS (LRFAB, DTT -DTE) m/z 441 [M + Hf.

D. Synthesis of Boc-(R.R)-Cyc(Ts)-Glv-OH

To a stirred solution of impure Boc-(R,R)-Cyc(Ts)-Gly-OMe prepared in Example 1C (217 g, 0.492 mol) in MeOH (1.05 L) was slowly added a 2.5 N solution of aqueous * NaOH (295 mL, 0.737 mol) and the resulting solution was stirred for 2 hours. The solvent was removed in vacuo and the residue was dissolved in H ₂ O (1.5 L). The solution was filtered to remove. a small amount of solid and washed with ether (7 x 1 L) to remove impurities (compound 1B), the combined filtrates were dried over MgSO 4 , and the solvent was removed in vacuo to leave a residue of 8.37 g. The pH of the aqueous solution was then adjusted to 2 with 1 N HCl and the product was extracted with ethyl acetate (3 x 1 L). The extracts were combined, washed with saturated NaCl (500 mL) and dried over MgSO 4 . The solvent was removed in vacuo and the residual ethyl acetate was removed by co-evaporation with ether (500 mL) and then CH ₂ Cl ₂ (500 mL) to give 205 g (97.6% yield) of the product as a white foam: ¹ H NMR (CDCl ₃ ) δ ._1,.15.-1.22.(m, 4H),-1.48-(s _f -9H),-1 _; 55-T.68 (m _? 3H), -2;í2-2.15-(m,· 1H) _l ''2;43'(s _l '3H), 3.41-3.49 (m, 2H), 3.97 (ABq, J = 17.9 Hz, Δν = 69.6 Hz, 2H), 4.79 (broad s, 1H), 7.31 (d, J = 8.3 Hz, 2H ), 7.77 (d, J = 8.3 Hz, 2H), 8.81 (broad s, 1H); MS (LRFAB, NBA - Li) m/z 443 [M + Li]*.

E. Synthesis of Boc-(R,R)-Cvc(Ts)-Glv-Glv-QEt

To Boc-(R,R)-cyc(Ts)-gly-OH (18.1 g, 43.1 mmol) in DMF (480 mL) was added HOBt. H ₂ O (7.92 g, 51.7 mmol) and EDC. HCl (9.91 g, 51.7 mmol) and the resulting mixture was stirred for 20 min at room temperature. GlyOEt was added to this solution. HCl (6.0 g, 43.1 mmol) and TEA (7.2 mL, 51.7 mmol) and the resulting mixture was then stirred for 16 h; 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 1N KHSO ₄ (250 mL), water (250 mL), saturated NaHCO ₃ (250 mL), and brine (250 mL) and dried (Na ₂ SO 4 ). Filtration and concentration afforded 21.9 g (99% yield) of pure product as a white foam: ¹ H NMR (DMSO-d _e ) δ 1.00 /1.10 (m, 1H), 1.19 (t, J = 7.6 Hz, 3H), 1.38 (s, 9H), 1.50-1.56 (m, 3H), 1.75-1.84 (m, 1H), 2.38 (s , 3H), 3.30-3-40 (broad s, 2H), 3.75-4.01 (complex m, 4H), 4.08 (q, J = 7.6 Hz, 2H), 6, 05 (broad s, 1H), 7.32 (d, J = 8.0 Hz, 2H), 7.77 (d, J = 8.0 Hz, 2H), 8.32 (t, J = 7, 2Hz, 1H); MS (HRFAB) m/z 518.2551 (M + Li)* ; 518.2512 calculated for C ₂ 4 H ₃₇ N ₃ O ₇ SLi.

F, Synthesis of the TFA salt Cvc(Ts)-Glv-Giv-OEt • 9 ···« * 9 9 9 999 9 *

999 9 9 9 9 ·

9 999999 99 99

To a solution of Boc-Cyc(Ts)-GJy-Gly-OEt (21.2 g, 41.4 mmol) in CH ₂ Cl ₂ (180 mL) was added TFA (44 mL) and the resulting mixture was stirred for 30 min at temperature. The solution was concentrated and the residue was dissolved in ether (50 mL) and precipitated in hexane (500 mL). The solvent was decanted and the residue was washed with 10:1 hexane/ether (500 mL). The resulting residue was properly dried under high vacuum to give 20.7 g (95% yield) of product as a tan foam: ¹ H NMR (DMS0-d ₆ ) δ 0.85 -0.96 (m, 1H), 1.031 .31 (complex m, 7H), 1.09 (t, J = 7.6 Hz, 3H), 2.00 (m, 1H), 2.39 (s, 3H), 3.02 (broad s, 1H), 7.41 (d, J = 8.1 Hz, 2H), 7.67 (d, J = 8.0 Hz, 2H), 8.25 (broad s, 3H), 9.09 (t , J = 5.63 Hz, 1H). MS (HRFAB) m/z 418.1990 (M -TFA + Li) ⁺ ; 418, 1988 calculated for C19H29N3O5S.

G. Synthesis of Boc-Om(Z)-Cvc(Ts)-Gly-Glv-OEt--------- -----”------ ' '

To Boc-Orn(Z)-OH (8.37 g, 22.8 mmol) in DMF (200 mL) was added HOBt. H ₂ O (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 room temperature. To this solution was added TFA salt Cys(Ts)-Gly-Gly-OEt (12.0 g, 22.8 mmol) and TEA (3.82 mL, 27.4 mmol) and stirring was continued for another 16 h. 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 1N KHSO ₄ (150 mL), water (150 mL), saturated NaHCO 3 ' (150 mL), and brine (150 mL) and dried (MgSO ₄ ). Filtration and concentration afforded 15.1 g (87% yield) of product as a white foam: ¹ H NMR (DMSO-de) δ 1.00 1.94 (complex m, 12 H), 1.15 (t, J = 7.4 Hz, 3H), 2.38 (s, H), 2.98 (broad s, 2H), 3.303.46 (m, 2H), 3.70-3.82 (m, 4H), 3.90-4.02 (m, 1H), 4.05 (t, J = 7.4 Hz, 2H), 5.00 (s, 2H), 6.43 (m, 1H), 7.17 (m, 1H), 7.20-737 (m, 8H), 7.87 (m, 2H), 8.30 (broad s, 1H); MS (LRFAB, NBA + HCl) m/z 760 (M + H) ⁺ .

H. Synthesis of the TFA salt Orn(Z)-Cvs(Ts)-Gly-Gly-OEt

To a solution of Boc-Orn(Z)-Cyc(Ts)-Gly-Gly-OEt (14.5 g, 19.1 mmol) in CH ₂ Cl ₂ (120 mL) was added TFA (30 mL) and the resulting mixture was stirred 30 minutes at room temperature. The solution was concentrated and the residue was dissolved in ether (100 mL). The ether was decanted, the residue dried under high vacuum to give 15.5 g (>100% yield containing TFA) of product as an orange foam: ¹ H NMR (DMSO-de) δ 0.97 -1.93 (complex m , 12H), 1.16 (t, J = 7.4 Hz, 3H), 2.38 (s, 3H), 2.98 (broad s, 2H), 3.31-3.50 (m, 2H ), 3.7136 ** « ··» ·»· · • · • 4 « 1

3.91 (m, 4H), 3.97-4.04 (m, 1H), 4.08 (q. J = 7.4 Hz, 2H), 5.00 (s, 2H), 7.23 -7.39 (m, 8H), 7.77-7.81 (m, 2H), 8.18 (broad s, 3H), 8.41 (broad s, 1H); MS (LRFAB, NBA + HCl) m/z 660 (M -TFA) ⁺ .

I. Synthesis of Boc-Gly-Orn(Z)-CvcfTs)-Gly-Gly-OEt

To a solution of Boc-Gly-OH (3.36 g, 19.2 mmol) in DMF (220 mL) was added HOBt.H ₂ O (3.52 g, 23.0 mmol) and EDC .HCl (4.41 g , 23.0 mmol) and the resulting solution was stirred at room temperature for 20 min. To this solution was added the TFA salt of Orn(Z)-Cyc(Ts)-GlyGly-OEt (14.8 g, 19.2 mmol). (3.20 mL, 23.0 mmol) and stirring was continued for an additional 12 h. The DMF was evaporated and the residue was partitioned between water (200 mL) and EtOAc (350 mL). The layers were washed with 1N KHSO ₄ ( 150 ml), water (150 ml), saturated NaHCO ₃ (150 ml), brine (150 ml) and dried (MgSO ₄ )? Filtration and concentration gave 13.7 g (87% yield) of pure product as a white foam: ¹ H NMR (DMSO-d _e ) δ 0.96 -1.10 (m, 1H), 1.17 (t, J = 7.4 Hz, 3H), 1.38 (s, 9H), 1.35-2.00 (complex m, 10H), 2.97 (m, 2H), 3.60 (broad s, 2H), 3.67-3.84 (m, 4H), 3 .93-4.03 (m, 3H), 4.06 (q, J = 7.4 Hz, 2H), 6.92 (broad s, 1Ή), 7.19 (m, 1H), 7.24 -7.37 (m, 7H), 7.60 (d, J = 8.3 hz, 1H), 7.76 (m, 2H), 7.38 (broad s, 1H).MS (LRFAB, NBA + Li) ⁺ m/z 823 (M + Li) ⁺ .

J. Synthesis of Boc-Gly-Orn(Z)-CvsfTs)-Gly-Glv-OH

To a solution of Boc-Gly-Orn(Z)-Cys(Ts)-Gly-Gly-OEt (13.3 g, 16.3 mmol) in methanol (100 mL) was added 1N NaOH (25 mL). The resulting mixture was stirred at room temperature and monitored by TLC. After 2 hours, 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 discarded. The pH was lowered to 3.5 with 1N KHSO ₄ and the aqueous phase was extracted with EtOAc (3 x 100 mL). The combined EtOAc layers were dried (MgSO ₄ ), filtered, concentrated to give 11.7 g (91% yield) of product as a white foam: ¹ H NMR (CDCb) δ 0.98 -1.25 (m, 2 H ), 1.38 (s, 9H), 1.40-1.92 (m, 10H), 2.38 (s, 3H), 2.97 (m, 2H), 3.62 (broad s, 2H), 3.75-3.85 (m, 3H), 2.97 (m, 2H), 3.62 (broad s, 2H), 3.753.85 (m, 3H), 3.95-4, 05 (m, 2H), 5.01 (s, 2H), 6.96 (broad s, 1H), 7.28 (m, 1H), 7.257.38 (m, 7H), 7.61 (d, J = 8.4 Hz, 1H), 7.78 (m, 2H), 8.25 (broad s, 1H).

K. synthesis of the TFA salt Gly-Orn(Z)-CysfTs)-Gly-Glv-OH «44« 4 4 4 4 4· v.

-2*7 · 4 «··· 4 4 44444* 4 j\J / · 4 4 4 4 444 . t • 4 4 444 «44 *44« •i’

To a solution of Boc-Gfy-Orn(Z)-Cys(Ts)-Gly-Gly-OH (11.2 g, 14.3 mmol) in CH ₂ C _{1 2} H (100 mL) was added TFA (24 mL) and the resulting solution was stirred for 30 minutes at room temperature

temperature. The solution was concentrated and triturated in ethyl ether (500 mL). Filtration gave 1

11.3 g (99% yield) of product as a white powder: ¹ H NMR (DMSO-d _s ) δ 0.95 1.98 (complex m, 12 H), 2.39 (s, 3H), 3, 01 (m, 2H), 3.38 (m, 1H), 3.65-4.10 (complex m,

7H), 4.18 (q, J = 7.4 Hz, 1H), 5.02 (s, 2H), 7.24-7.40 (m, 9H), 7.77-7.85 (m , 2H), 8.13 (broad s, 1H), 8.42 (d, J = 8.3 Hz, 1H); MS (HRFAB) 689.2953 (M - TFA ) ⁺ ; 689.2969 calculated for C32H45N6O9S.

and

L. Synthesis of cyclo -(Glv-Orn(Z)-Cvs(Ts) -Glv-Glv-)

A TFA solution of the Gly-Orn(Z)-Cys(Ts)-Gly-Gly-OH salt (5.0 g, 6.23 mmol) in dry .......degassed-DMF (1520 mL) was treated with TEA-(4.-74 mL; 12.5 rnmol) 'and cooled to -......

40°C. DPPA (1.64 mL, 7.60 mmol) was added dropwise over 10 min and the reaction was then stirred at -40 °C for 3 h. The reaction was then placed in a -2°C bath and allowed to stand at this temperature for 16 hours. Water (1520 mL) was added and the resulting solution was stirred with a mixture of ion exchange resin beads (750 g) for 1 h at room temperature. The resin was filtered off and the solution was concentrated to a volume of approximately 100 mL (DMF). Addition of ethyl ether (500 mL) gave a solid residue, which was dissolved in methanol (100 mL) and precipitated again by addition of ethyl ether (500 mL). Filtration afforded 3.26 g (78% yield) of product as a white powder: 1H NMR (CDCb) δ 0.96 -2.10 (complex m, 14 H), 2.37 (broad s, 3H), 2.68-3.05 (m, 3H), 3.423.90 (complex m, 8H), 4.14 (m, 1H), 4.20 (m, 1H), 4.97-5.08 (m , 3H), 6.42 (d, J = 8.4 Hz, 1H), 7.20-7.39 (m, 7H), 7.65-7.78 (m, 2H), 9.15 ( wide s, 1H), 9.22 (wide s, 1H);

MS (HRFAB) m/z 671.2842 (Μ + H ) ⁺ ; 671.2863 calculated for C ₃₂ H ₄₃ N ₆ O ₈ S.

M. Synthesis of cy/r/o-øGlv-Orn-Cys(Ts)-Gly-Glv-)

To a solution of cyclo -(Gly-Orn(Z)-Cys(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 then cooled. The mixture was filtered under argon through a Celt pad, the filtrate was concentrated to give 2.86 g (89% yield) of product as a white foam: ¹ H NMR (DMSO -d ₆ ) δ 0.94 -2.22 (complex m, 12H), 2.39 (s, 3H), 2.55-2.95 (m, 7H), 3.42-3.89 (complex m, 9H), 4.11 (m, 1H ), 4.39 (m, 1H), 6.43 (d, J = 8.4 Hz,

JO · * ···· · * Φ Φ ΦΦΦ « «

·. · · · φ « φ I ·· φ φφφ φφφ ·Φ

1Η), 7.27 (d, J = 9.3 Hz, 1Η), 7.25-7.45 (m, 2Η), 7.64-7.80 (m, 2Η), 9.12- 9.29 (m, 2H); MS (HRFAB) m/z 537.2511 (M + H) ⁺ ; 537.2495 calculated for C^HseNeSOe.

N. Synthesis of cyclo-(Gly-Om(lithocholvl)-Cvs(Ts)-Gly-Gly-)

To a solution of cyWo-fGly-Orn-Cys(Ts)-Gly-Gly-) (1.0 g, 1.9 mmol) in CHClb (25 mL) was added NHS active lithocholic acid ester (881 mg, 1.9 mmol ) and the resulting mixture was then stirred for 16 hours. Addition of ethyl ester (50 mL) gave a solid. Filtration afforded 946 mg (56% yield) of the product as a tan powder; 'H NMR (CD ₃ OD) δ 0.66 (m, 3H), 0.93 (broad s, 6H), 0.94-2.37 (complex m, 48H), 2.43 (s, 3H) , 2.80-4.60 (broad m, 14H), 7.39 (broad s, 2H), 7.80 (broad s, 2H); MS (HRFAB) m/z 895.5432 (Μ + H ) ⁺ ; 895.5367 calculated for C4eH ₇ 5N ₆ O _fl S.

O. Synthesis of 2.3-(R.R)-cyclohexane-6-(SH3-(lithochoiylamino)propylM,4,7.10.13-pentaazcyclopentadecane

To a suspension of cyWo-fGly-Orn(lithocholyl)-Cys(Ts)-Gly-Gly-) (2.70 g, 3.00 mmol) in THF (50 mL) was added lithium aluminum hydride (51.0 mL and 0. 1 M solution). The resulting mixture was subsequently refluxed for 16 hours. The reaction mixture was cooled to -20°C and quenched (carefully) with 5% Na ₂ SO ₄ (30 mL) followed by methanol (30 mL). This solution was stirred for 1 hour at room temperature and concentrated to a dry powder. The powder was suspended in ethyl ether (3 x 200 ml) and filtered. The ether was concentrated and the oil recrystallized from acetonitrile to give 800 mg (40% yield) of product as a colorless oil: ¹ H NMR (C6D6) δ 0.64 (s, 3H), 0.67 (s, 3H), 0 .88 (d, J = 3.0 Hz, 3H), 0.84-2.61 (complex m, 52 H), 2.38-2.95 (complex m, 14 H), 3.46 (m , 3H); ¹³ C NMR (CDCl 3 ) δ 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 , 23.8, 20.8, 18.6, 12.0; MS (LRFAB, NBA + Li) m/z 677 (M + Li) ⁺ .

P, Synthesis of idichloro 2,3-(R,R)-cyclohexane-6-(S)-[3-(lithocholvamino)propyl-1,4,7,10.13penta-azaccyclopentadecane manganatehol

2,3-(R,R)-cyclohexane-6-(S)-{3-(lithocholyamino)propyl}-1,4,7,10,13-pentazacyclopentadecane prepared in Example 10 (547 mg, 0.817 mmol) was added to hot39 • »· » » » ....

• · ··· · ' * * ΚΙ , _ν ··· · · * * * ·' * *· ·* a· a* to an anhydrous methanol solution (50 mL) containing manganese chloride (103 mg, 0.818 mmol) in dry nitrogen atmosphere. After refluxing for 2 hours, the solution was reduced to a dry residue which was dissolved in a solvent mixture of THE (35 mL) and ethyl ether (5 mL) and filtered through a celite pad. Concentration and trituration in ethyl ether gave, after filtration, 512 mg (79% yield) of the complex as a white solid: FAB mass spectrum (NBA) m/z 760 [M-Clf; Analysis calcd for C 2 HraNeoMnCt C 61.79; H, 9.87; N, 10.55; Cl, 8.90. Actual values: C, 62.67; H, 9.84; N, 8.04; Cl, 8.29.

Example 2

Kinetic analysis of stopped flow

Stopped-flow kinetic analysis was used to determine whether a compound can catalyze superoxide dismutation (Riley, DP, Rivers, WJ, and Weiss, RH, StoppedFlow Kinetic Analysis for Monitoring Superoxide Decay in Aqueous Systems,” Anal. Biochem. 196. 344-349 [1991]). To achieve consistent and accurate measurements, all reagents were biologically pure and metal-free. To this end, all buffers (Cabiochem) were biologically screened to be free of metals and were handled with labware that was washed before use first in 0.1 N HCl, then in purified water, then in a 10 ⁴ M EDTA bath at pH 8, and then washed in purified water and dried for several hours at 65°C. An anhydrous solution of potassium superoxide in DMSO (Aldrich) was prepared under a dry, inert atmosphere of argon in a vacuum glovebox atmosphere using dry glassware. The DMSO solution was prepared fresh before each stopped-flow experiment. A pestle and mortar was used to crush the yellow solid of potassium superoxide (approximately 100 mg). The powder was then dissolved in a few drops of DMSO and the suspension was placed in a container containing another 25 ml of DMSO. The resulting suspension was stirred for 1/2 hour and then filtered. This procedure is reproducible at approximately 2 mM concentrations of superoxide in DMSO. These solutions were transferred to a glove bag in sealed tubes before connecting the syringe under a nitrogen atmosphere. It should be noted that DMSO/superoxide solutions are very sensitive to water, heat, air and external metals. The fresh, clear solution has a faint yellowish color. Water for the buffer solutions was used from an in-house Bamstead Nanopure Ultrapure Séries 550 deionized water system and then distilled twice, first from alkaline manganese · * ···· ♦ · · · ·· · · ·

444 4 4 · · 4 • 4 4 4 · · 444 44 44 of potassium, and then from a dilute EDTA solution. For example, a solution containing 1.0 g of potassium permanganate per 2 liters of water, plus the sodium hydroxide needed to adjust the pH to 9.0, was added to a 2 liter vessel with a top section adjusted for solvent distillation. This distillation oxidized any trace of the organic compound in the water. The final distillation was carried out in a nitrogen atmosphere in a 2.5 liter vessel containing 1500 ml of water from the previous distillation and 1.5 χ 10 ⁶ M EDTA. This step removed the remaining traces of metals from the ultrapure water. To prevent loss of EDTA from the entire volume from the reflux arm to the solid top, 40 cm of the vertical arm was wrapped with glass beads and sealed with insulation. This system produced deaerated water that could be measured with a conductivity of less than 2.0 nanoohms/cm ²

The stopped flow spectrometer system was designed and manufactured by Kinetic. Instruments lne. (Ann Arbor, Ml) and was connected to a MAC IICX personal computer. Stopped flow analysis software has been tested by Kinetic Instruments lne. and written in QuckBasic with the MacAdios driver. A common injection volume (0.10 ml buffer and 0.006 ml DMSO) was calibrated due to the large excess of water in the DMSO mixture. The current ratio was approximately 19/1, therefore the initial concentration of superoxide in the aqueous solution was in the range of 60-120 μΜ. Since the absorption coefficient of superoxide in H ₂ O reported in the literature at 245 nm is approximately 2250 M' ¹ cm' ¹ (1), an initial absorbance value of approximately 0.3-0.5 was expected for a 2 cm cuvette measuring path , which was also confirmed experimentally. Aqueous solutions mixed with DMSO superoxide solution were prepared using 80 mM concentration of Heppes buffer, pH 8.1 (free acid + Na form). One of the stock syringes was filled with 5 ml of DMSO solution, while another was filled with 5 ml of aqueous buffer solution. The inlet block for sample injection, the mixing chamber and the spectrometric cell were pre-tempered in a circulating thermostatic water bath at a temperature of 21.0 ± 0.5 °C.

Before starting data collection for superoxide breakdown, a baseline average was obtained by several injections of buffer and DMSO solution into the mixing chamber. For these injections, the average was recorded and kept as a baseline. The first injections obtained during the series of measurements were aqueous solutions that did not contain catalyst. This confirmed that neither series of measurements contained contaminants that could be formed by the initiation of superoxide decomposition. If superoxide decomposition occurred in injections with the buffer solution second in order, the solution used may have been a manganese complex. The potential of SOD was generally determined for a large concentration range. Since the initial con41 · · ··♦· · · · · ♦·* · . · · · · ·· ·· · 1 ·· ♦ ··· ··♦ *· ·· centration of superoxide after mixing DMSO with an aqueous buffer of approximately 1.2 χ 1(Γ* M, it was necessary to use a concentration of manganese complex which was at least ²⁰ times lower than the concentration of superoxide Cricket Graph) to perform a standard kinetic analysis. The catalytic rate constant for the dismutation of the manganese complex from Example 1 was determined from the linear dependence of the obtained rate constants (Κ,^) on the concentration of the manganese j> complex. The values of K^ were obtained from the linear dependence The absorbance at 245 nm of the manganese complex dismutation time (M' ¹ s' ¹ ) of the manganese complex at pH = 8.1 and 21°C was found to be 0.77 χ 10* ⁷ M ¹ s '\ <

.. The nitrogen-containing macrocyclic ligand-manganate complex ^of Example 1 is an effective catalyst for dismutation of superoxide, as is evident from kio, above.

Claims (18)

PATENT CLAIMS A complex compound of the general formula wherein R, R, R 1, R 1. _R2, R2. R 3, R ₃ , R ₄ , R 41 R 5, R 5, R 6, R 6! R7, Rz, Rej Re! R 9 and R 8 are independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylcycloalkyl, cycloalkenylalkyl, alkylcycloalkyl, alkenylcycloalkyl, alkylcycloalkenyl, alkenylcycloalkenyl, heterocyclic, aryl and aralkyl amino and α-alkyl radicals; or R 1 or R ₂ and R ₂ or R ₂ , R ₃ or R ₃ and R ₄ or R ₄ , R ₅ or R ₅ and Re or Re, R ₇ or R ₇ and Re or Re and Re or R 8 and R or R together with the carbon atoms to which they are independently attached to form a saturated, partially saturated or unsaturated ring having from 3 to 20 carbon atoms; or R or R and R 1 or R 1 and R ₂ or R _{2 |} R ₃ or R ₃ and R ₄ or R ₄ , R 5 or R ₅ and R e or R e, R ₇ or R ₇ and R e or R e and R ₉ or R 8 together with the carbon atoms to which they are independently attached form a heterocycle having 2 up to 20 carbon atoms, provided that when the nitrogen-containing heterocycle is an aromatic heterocycle that does not contain hydrogen attached to the nitrogen, the hydrogen attached to the nitrogen of said formula, that nitrogen is also present in the macrocycle and R groups attached to the same carbon atoms of the macrocycle; and combinations thereof; wherein (1) one to five R "groups are attached to the biomolecule by a linking group, (2) one of X, Y and Z is attached to the biomolecule by a linking group, or (3) one to five" R "groups and one of X, Y and Z are attached to the biomolecule via a linking group; and the biomolecules are independently selected from the group consisting of steroids, bicarbonates, fatty acids, amino acids, peptides, proteins, antibodies, vitamins, - - - - - - - - - - - - - - - - - - - 43 · Lipids, phospholipids, phosphates, phosphonates, nucleic acids, enzymatic enzymes substrates, enzyme inhibitors and enzyme receptor substrates and the linking group are derived from a substituent attached to the R 'group or said X, Y and Z, which is reactive with a biomolecule, and is selected from the group consisting of -NH ₂ , -NHR ₁₀ , - SH, -OH, -COOH, -COOR _10, -CONH _2, -NCO, -NCS, -COOX ", alkenyl material, alkynyl, halide, p -toluenesulfonate, methanesulfonate, tresylate, triflate and phenol, wherein R is alkyl, aryl, or alkaryl and X 'is a halide; wherein M is Mn or Fe and wherein X, Y and Z are ligands independently selected from the group consisting of halide, oxo, aquo, hydroxo, alcohol, phenol, oxygenate, peroxo, hydroperoxo, alkylperoxo, arylperoxo, ammonia, alkylamino, arylamino, heterocycloalkylamino , heterocycloarylamino, amine oxide, hydrazine, alkylhydrazine, arylhydrazine, nitric oxide, cyanide, cyariate, thiocyanate, isocyanate, isothiocyanate, alkylnitrile, - aryinitrile, -alkyl isonitryl, - arylisonitrile, -nitrate, - nitrite, ... ..... alkylsulfonic acids, ary Isulfonic acids, alkylsulfoxide, arylsulfoxide, alkylarylsulfoxide, alkylsulfenic acids, arylsulfenic acids, alkylsulfinic acids, arylsulfinic acids, alkylthiol carboxylic acids, arylthiol carboxylic acids, alkylthiolthiocarboxylic acids, arylthiolthiocarboxylic acids, arylthiolthiocarboxylic acids alkylaryl ureas, thioureas, arylthioureas, alkylarylthioureas, sulphate, sulphite, disulphite, disulphite, thiosulphate, thiosulphite, hydrogen sulphite, alkylphosphine, arylphosphine, alkylphosphine oxide, arylphosphine oxide, alkylphosphine sulphide, alkylphosphine sulphide, alkylphosphine sulphide, alkylphosphine sulphide, alkylphosphine sulphide, thiophosphate, phosphite, pyrophosphite, triphosphate, hydrogen phosphate, dihydrogen phosphate, alkylquanidine,. arylquanidinu, alkylarylquanidinu, alkyl carbamate, arylcarbamate, alkylarylkarbamátu, aryldithiokarbamátu, alkylaryldithiokarbamátu, bicarbonate, carbonate, perchlorate, chlorate, chlorite, hypochlorite, perbromate, bromečnanu, bromite, hypobromite, tetrahalogenmangananu, tetraflourborátu, hexafluorophosphate, hexafluoroantimonate, hypophosphite, iodate, periodate, metaborate, tetraarylborate, tetraalkylborate, tartrate, oxalate, succinate, citrate, ascorbate, saccharinate, amino acid, hydroxamic acid, thioparatoluene sulfonate, and ion exchange resin anions, or their corresponding anions, or X, Y and Z are independently attached to one or more R 'groups, where n is 0 or 1. · · • · • • · · · · · · · The compound of claim 1, wherein 1 to 2 of the R 1 groups are attached to the biomolecule by a linking group and X, Y or Z are not attached to the biomolecule by a linking group. The compound of claim 1, wherein one of X, Y and Z is attached to the biomolecule by a linking group and no R group is attached to the biomolecule by a linking group. The compound of claim 1, wherein at most one Έ "moiety, attached to the carbon atom of the macrocycle located between the nitrogen atoms, has a biomolecule attached via a linking group. The compound of claim 1, wherein at least one of the "R groups, in addition to the" R "groups that are attached to the biomolecule via a linking group, is selected from the group consisting of alkyl, cycloalkyl, cycloalkylalkyl, aralkyl, alkaryl, aryl, heterocycles and residues J attached to α-carbons of α-amino acids and the remaining ⁿ R 'groups are independently selected from the group of hydrogen, saturated, partially saturated or unsaturated cycles or nitrogen-containing heterocycles. The compound of claim 5, wherein at least two of the "R" groups, in addition to the "R" groups, which are attached to the biomolecule via a linking group are selected from the group consisting of alkyl, cycloalkyl, cycloalkylalkyl, aralkyl, alkaryl, aryl, heterocycles and residues attached to α-carbons of α-amino acids. The compound of claim 5, wherein at least one of the "R groups", except for the "R" groups that are attached to the biomolecule via a linking group is alkyl, and the remaining R "groups are independently selected from hydrogen, saturated, partially saturated or unsaturated cycles. 8. A compound according to claim 1, wherein at least one of R or R / and R ₂ or R _{2 l} R ₃ or R ₃ and R ₄ or R 4, R or R and R or R, R ₇ or R ₇ and Re, or Re and R ₉ or R 8 'and R 8 or R 8 together with the carbon atoms to which they are attached represent a saturated, partially saturated or unsaturated cycle of 3 to 20 carbon atoms and the remaining R groups, except for the "R groups" attached to the biomolecule via bonding groups - -, -w V w v., _ ···· · * ··· The compounds are independently selected from hydrogen, nitrogen-containing heterocycles or alkyl groups. 9. A compound according to claim 8, wherein at least two of Ri or R _t and R ₂ or R _2, R 3 or R ₃ and Ft, or R 4, R ₅ or R and R or R, _R? or R? and R e or R e and R ₉ or R ₉ 'and R or R together with the carbon atoms to which they are attached represent a saturated, partially saturated or unsaturated cycle of 3 to 20 carbon atoms and the remaining groups ^B R ", except"R' The groups which are attached to the biomolecule by a linking group are independently selected from hydrogen, nitrogen-containing heterocycles or alkyl groups. The compound according to claim 8, where. said saturated partially-saturated or ... unsaturated cycle is cyclohexyl. * The compound of claim 10, wherein said remaining "R" groups, in addition to the R groups, I. which are attached to the biomolecule by means of linking groups are independently selected from hydrogen or alkyl groups. A compound according to claim 1, wherein said R or R and R 1 or R ₂ and R ₂ or R ₂ , R 3 or R ₃ and R 4 or R 4, R 5 or R ₅ and R ₆ or R ₇ , R ₇ or R ₇ or R ₇ ; and R e or R e and R ₉ or R ₉ together with the carbon atoms to which they are attached form a nitrogen-containing heterocycle of 2 to 20 carbon atoms and the remaining "R" groups in addition to the "R" groups that are attached to the biomolecule via The alkyl groups are independently selected from hydrogen, saturated, partially saturated or unsaturated cycles or alkyl groups. The compound of claim 1, wherein X, Y and Z are independently selected from the group consisting of anions of halide, organic acid, nitrate, and bicarbonate. The compound of claim 1, wherein M is Fe. The compound of claim 1, wherein M is Mn. A pharmaceutical composition in unit dosage form, useful for dismutering superoxide, comprising (a) a therapeutically or prophylactically effective amount of the complex of claim 1, and (b) a non-toxic, pharmaceutically, acceptable carrier, adjuvant or admixture. A method of preventing or treating a disease or disorder that is caused, at least in part, by superoxide, comprising administering to a patient as a prevention or treatment a therapeutically, prophylactically, pathologically or resuscitatively effective amount of a complex of claim 1. The method of claim 17, wherein said disease or disorder belongs to the group consisting of ischemic reperfusion injury, surgery-induced ischemia, inflammatory bowel disease, rheumatoid arthritis, atherosclerosis, thrombosis, platelet aggregation, oxidation-induced damage and tissue disorders, osteoartriti · ^'5 * dy, psoriasis, rejection of a transplanted organ disorders caused by radioactive záře-,' him, stroke, acute pancreatitis, diabetes mellitus dependent inzuliriu, respiratory insufficiency in adults and children, metastasis and carcinogenesis. The method of claim 18, wherein said disease or disorder belongs to the group comprising ischemic reperfusion injury, stroke, atherosclerosis, and inflammation. inflammatory bowel disease.