CZ39898A3 - Bioconjugates of macrocyclic ligand manganese complexes containing nitrogen and being active as catalysts of 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 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

Bioconjugates of manganese nitrogen-containing macrocyclic ligand complexes effective as catalysts for superoxide dismutation

Technical field

The invention relates to compounds effective as superoxide dismutation catalysts. The invention relates to manganese (II) and manganese (III) complexes of 15-membered macrocyclic ligands that catalytically dismutal superoxide. In another aspect, the invention relates to manganese complexes with nitrogen-containing 15-membered macrocyclic ligands that are conjugated to targeted biomolecules.

BACKGROUND OF THE INVENTION

The superoxide dismutase enzyme catalyses the conversion of superoxide to oxygen and hydrogen peroxide according to Equation (1) ° 2 ⁺ ° 2 - ^{+ 2H +} - ° 2 + H ₂ O _: (hereinafter referred to as dismutation). Reactive oxygen metabolites derived from superoxide are believed to contribute to tissue pathology in many inflammatory diseases and disorders such as reperfusion injury to ischemic heart muscle, inflammatory disease of the internal organs, rheumatoid arthritis, osteoarthritis, atherosclerosis, hypertension, methertension, methertension organ transplants, radiation damage, asthma, influenza, stroke, burns and trauma. See, for example, Bulkley, GB, Reactive oxygen metabolites and reperfusion injury, aberrant activation of reticuloendothelial function, The Lancet, Vol. 344, pp. 934-36, October 1, 1994; Grisham, MB, Oxidants and Free Radicals in Inflammatory Diseases of Internal Organs, The Lancet, Vol. 344, pp. 859-861, September 24, 1994; Cross, CE et al., Reactive Oxygen Particles and Lungs, The Lancet, Vol. 344, pp. 930-33, October 1, 1994; Jenner, P., Oxidative Damage in Neurodegenerative Disease, The Lancet, Vol. 344, pp. 796-798.17. September, 1994; Cerutti, PA, Oxy-Radicals and Cancer, The Lancet, Vol. 344, pp. 862-863, September 24, 1994; Simic, MG, 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 (1991); Petkau, A., Cancer Treat. Roar. 13, 17 (1986); McCord, J., Free Radicals, Biol. Med., 2, 307 (1986); and Bannister, JV, et al., Crit. Roar. Biochem., 22,111, (1987), The Lancet references above postulate • 4 ·· • 4 4 I

4 4 • 4

4445 44 · 4 ··

4 4 4

4 4 4

444 * 4 * 44

4 4

4 4 4 4 The link between superoxide free radicals and a number of diseases. In particular, references by Bulkley and Grisham emphasize that there is a link between dismutal superoxide and the ultimate treatment of the disease.

It is also known that superoxide is involved in endothelial vascular release factor (EDRF) failure that has been identified as nitric oxide (NO) and that EDRF is protected from superoxide dismutase defect. This suggests a central role of activated superoxide oxygenated particles in the pathogenesis of vascular spasm, thrombosis and atherosclerosis. See, for example, Gryglewski, R.J., et al., "Superoxide Anion Involved in Defect of Endothelial Vascular Release Factor", Nature, Vol. 320, pp. 454-56 (1986) and Palmer, R.M.J., et al., "Nitric oxide release is responsible for the biological activity of endothelial releasing factor", Nature, Vol. 327, pp. 523-26 (1987).

Clinical trials and studies in animals with congenital, recombinant, and modified modified superoxide dismutase enzymes have been conducted or are ongoing to demonstrate the therapeutic efficacy of superoxide reduction in the aforementioned disease states. Numerous probes have arisen using enzymes as potential therapeutic agents, including loss of oral activity, short half-life in vivo, immunogenicity of enzymes obtained outside the human body, and poor tissue distribution.

Manganese complexes of 15-member nitrogen-containing macrocyclic ligands that are low molecular weight superoxide dismutase (SOD) mimics are useful as therapeutic agents and avoid many of the problems associated with SOD enzymes. However, it would be desirable to direct imitations of SOD to the desired target in the body where the compound can be concentrated for optimal effect. Without any way of rendering the compounds "targeted", increased doses are sometimes necessary to achieve an effective concentration at the site of interest. These increased doses can sometimes result in undesirable side effects on the patient.

It has now been found that the macrocycles or manganese complexes of the invention can be attached, ie conjugated, to one or more targeted distribution molecules via a linking group to form a targeted biomolecule-targeted distribution - a macrocycle or a targeted biomolecular-conjugate-manganese complex.

• · 4

4 4 · 4 4 4

4 «4 4,444 4,444

444 4,444 4,444 · 4

444444 444

444445 44 4 4 «··

SUMMARY OF THE INVENTION

It is an object of the invention to provide bioconjugates of manganese (II) or manganese (III) nitrogen-containing 15-membered macrocyclic ligand complexes which are low molecular weight superoxide dismutase (SOD) mimics useful as therapeutic agents for inflammatory disease states or disorders mediated, at least in part, by superoxide. . It is another object of the invention to provide bioconjugates of manganese (II) complexes of nitrogen containing 15-membered macrocyclic ligands which are useful as magnetic resonance imaging (MRI) contrast agents with improved kinetic stability, increased oxidative stability and improved hydrogen bonding capability. It is another object of the present invention to provide bioconjugates of manganese complexes of nitrogen containing 15-membered macrocyclic ligands that can be targeted to a specific site in the body. . ____. ___,

According to the invention there are provided bioconjugates of manganese (II) and manganese (III) complexes of 15-membered cyclic cyclo -articles containing more severe (71) members, with up to five "R" groups attached to the biomolecules via a linker, (2) one of X, Y and Z is attached a linker group, or (3) one to five "R" groups and one of X, Y and Z are attached to the biomolecules via a linker group; and the 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 ^u group or X, Y and Z which is reactive with the biomolecule and is selected from the group consisting of -NH _2, -NHR _10, -SH, -OH, -COOH, -COOR, - CONH ₂ , -NCO, -NCS, COOX ", alkenyl, alkynyl, halide, p-toluenesulfonate (tosylate), methanesulfonate, tresylate, triflate and phenol, wherein R _w is alkyl, aryl, or alkylaryl and X" is a halide.

The present invention is directed to bioconjugates of manganese (II) or manganese (III) complexes of nitrogen containing 15-membered macrocyclic ligands that catalyze the conversion of superoxide to oxygen and hydrogen peroxide. These complexes can be represented by formula (I);

• 9 • 99 «« 9

where R, R, R 1, R 1, R 2, R 2, R 3, R 3, R 4, R 4, R 5, R 5, R 8, R ₈ , R ₇ , R ₇ , R 8, R ' _{8 |} R _g and R ' _g independently represent hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylcycloalkyl, cycloalkenylalkyl, alkylcycloalkyl, alkenylcycloalkyl, alkylcycloalkenyl, alkenylcycloalkenyl, heterocycle, aryl and aralkyl radicals, and radicals attached to α-α-carbon ; or 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 ₁₅ and -R 6 - or-R- ₆ , -R ₇ -or-Rf ₇ -a-R8- or -R-8-R9- or -R-ga-R- or-R-together with the carbon atoms to which they are attached independently to form saturated, partially saturated or unsaturated cycles having 3 to 20 carbon atoms ; or R or R 'and R' or R ' _b R ₂ or R' ₂ and R 3 or R ' ₃ , R 4 or R' ₄ and R ₅ or R ' ₅ , R e or R' _s and R ₇ or R ' ₇ , and R e or R ' ₈ and R g or R' _g together with the carbon atoms to which they are attached independently to form a nitrogen-containing heterocycle of 2 to 20 carbon atoms, provided that when the nitrogen-containing heterocycle is an aromatic heterocycle which does not contain hydrogen bonded to nitrogen hydrogen bonded to the nitrogen of said formula (I), the nitrogen of which is also present 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 "R" groups are attached to the biomolecules 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 are attached to the biomolecules through a linking group; and the 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 moiety from a substituent attached to the "R" group or to X, Y and Z which reacts with the biomolecule and is selected from the group consisting of -NH ₂ , -NHR 10, -SH, -OH, -COOH, -COORw, -CONH ₂ , -NCO, 5 * * »· 9 9 9 9 9 · · •

Η · · * * «« «« «» »» »» «

COOX ', alkenyl, alkynyl, halide, p-toluenesulfonate, methanesulfonate, tresylate, triflate and phenol, wherein R ₁₀ is alkyl, aryl, or alkylaryl and X "is a halide.

X, Y and Z represent suitable charge neutralizing ligands or anions that are derived from a monodentate or polydentate coordinating ligand or ligand system or a corresponding anion (e.g., 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 halides, oxo, aqua, hydroxo, alcohol, phenol, molecular oxygen, peroxo, hydroperoxo, alkylperoxo, arylperoxo, amine, alkylamine, arylamine, heterocycloalkylamine, heterocycloarylamine, amine oxides, hydrazine, alkylhydrazine, arylhydrazine, nitric oxide, cyano, cyanato, thiocyanato, isocyanato, isothiocyanato, alkylnitrile, arylnitrile, nitrato, nitrito, azido, alkylsulfonic acid, arylsulfonic acid, alkylsulfoxide, arylsulfoxide, alkylarylsulfoxide, alkylsulphine sulfoxide, alkylsulphine sulfoxide, alkylsulphine sulfoxide arylthiolcarboxylic acid, alkylthiolthiocarbodoxylic acids, arylthiololthiocarboxylic acid, alkylcarboxylic acid (e.g. acetic acid, trifluoroacetic acid, oxalic acid), arylcarboxylic acid (e.g. benzoic acid, phthalic acid), urea, urea alkyl, aryl urea, al. kylarylmočovina, thiourea, alkylthiomočovina, arylthiomočovina, alkylarylthiomočovina, sulfate, sulfite, bisulfate, bisulfite, thiosulfate, thiosulfite, hydrosulfite, alkyl phosphine, aryl phosphine, alkyl phosphine oxides, arylfosfinoxid, alkylarylfosfinoxid, alkylfosfinsulfid, arylfosfinsulfid, alkylarylfosfinsulfid, alkylphosphonic acid, alkylphosphonic acid, Alkyl phosphonium acid arylphosphine acid diphenylphosphinite acid arylfosfinitá acids, phosphates, thiophosphates, phosphonate, diphosphonate, triphosphate, hydrogen phosphate, dihydrogenphosphate, alkylquanidine, arylguanidino, alkylarylguanidino, alkylcarbamate, arylcarbamates, alkylaryl carbamates, alkylthiocarbamates, arylthiokarbamat, alkylarylthiokarbamat, alkyldithiocarbamates, aryldithiokarbamat, álkylaryldithiokarbamat, hydrogen carbonate, perchlorate, chlorate, chlorite, hypochlorite, bromate, bromate, bromite, bromate, tetrahalomanganese, tetrafluoroborite an, hexafluorophosphate, hexafluoroantimonate, hypophosphite, iodate, periodate, metaborate, tetraarylborate, tetraalkylborate, tartrate, salicylate, succinate, citrate, ascorbate, saccharinate, amino acid, hydroxamic acid, thiotosylate, and an ion of one or more ion exchange, of X, Y and Z are independently attached to one or more "groups R ⁱⁿ which n is O or 1. more preferred ligands ·« «« · «· ·· · · *« ·· · »···· * · · A a * * * * nichž nichž nichž * * nichž * nichž nichž nichž nichž nichž nichž nichž nichž nichž nichž nichž nichž nichž X, Y and Z are selected, including anions of halides, organic acids, nitrate and bicarbonate,

Linking groups, also referred to herein as "linker", are derived from specific functional groups attached to "R groups" or to X, Y, and Z, and have the function of linking the biomolecule to "R" groups or X, Y, and Z. selected from the group consisting of -NH ₂ , -NHR _W , -SH, -OH, -COOH, -COOR 10, -CONH ₂ , *

NCO, -NCS, -COOX, alkenyl, alkynyl, halide, p-toluenesulfonate, methanesulfonate, tresylate, triflate and phenol, wherein R _w is alkyl, aryl, or alkylaryl and X 1 is a halide. Usually the more preferred alkenyl group is ethenyl and the more preferred alkynyl group is ethinyl. Functional groups on the "R" group or X, Y and Z are capable of reacting with a biomolecule, ie, reacting with a functional group on steroids, carbohydrates, fatty acids, amino acids, peptides, proteins, antibodies, vitamins, lipids, phospholipids, phosphates, phosphonates, nucleic acids. enzyme substrates, enzyme inhibitors, enzyme receptor substrates, and other biomolecules used with a targeted ^_ distribution. ^{If a} ^7R Kup moiety or X, Y and Z reacts with a biomolecule, the functional group is modified and this derived functional group is hyphen. For example, if the -NH ₂ functional group attached to the "R" group reacts with a steroid such as in Example 1, the linker is -NH-. The precise structure of the specific linker groups will be readily apparent to those skilled in the art and will depend upon the specific functional group and btomolecule selected. The specific reaction conditions for the reaction of a functional group attached to the "R" groups or X, Y and Z with a biomolecule will be readily apparent to those skilled in the art.

A functional group useful for forming a linker, defined herein as a "linker precursor", may be present on the "R" groups at the time the macrocycle is being prepared or may be added or modified after the preparation of the macrocycle or its manganese complex. Similarly, the linker precursor may be present on the axial ligand, i.e., X, Y or Z, when the manganese complex is prepared or the axial ligand exchange reaction is performed to exchange the axial ligands present in the manganese complex.

The macrocycle of the invention may be complexed with manganese both before and after conjugation to a targeted biomolecule depending on the specificity of the biomolecule utilized. A conjugate of a macrocyclic complex and a biomolecule with targeted distribution is defined herein as a "bioconjugate".

• 9

9 «99 99 · * 9 9 9 9 9 9 9 9

9,999,999

9 »« 98 9

9 99 99 99 9

9 9 9

9 ··

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99

Targeted drug distribution is well known to those skilled in the art. See, for example, J. A. Katzenellenbogen et al., Journal of Nuclear Median, Vol. 33, No. 4, 1992,558, and J. A. Katzenellenbogen et al., Bioconjugate Chemistry, 1991, 2, 353. Targeted distribution agents are typically biomolecules. Biomolecules of the invention are biologically active molecules that are site specific, i.e., are known to concentrate in a particular important organ or tissue. Biomolecules are selected to direct the tissue distribution of the bioconjugate through receptor binding, association with membranes, solubility in membranes, and the like. Such 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. Biomolecules also include those biomolecules that are a combination of the above biomolecules, such as a combination of a steroid with a carbohydrate, for example digitonin.

Specific ^{_ _} biomoíekulý7ktěřé mohoiri3ýt'využitý '} Fčílění'člopožadóvářréhoořgaňu' or tissue are known in the art or are skilled in the art readily apparent. The biomolecules of the invention are commercially available or can be readily prepared by a skilled artisan using conventional methods.

Normally, it is preferable that at most one "R" group attached to the carbon atoms located between the nitrogen atoms in the macrocycle has the biomolecule attached via a linker. In addition, more preferred compounds are those having one to five, more preferably one to two, "R" groups attached to the biomolecule and none of the groups X, Y and Z are attached to the biomolecule, or those having one of the X groups, Y and Z bound to the biomolecule and none of the "R" groups are bound to the biomolecule.

At the same time, more preferred compounds are those wherein at least one, more preferably at least two, of "R" ^! groups, in addition to the "R" groups that are attached to the biomolecule, represent the hydrocarbon radicals alkyl, cycloalkylalkyl and aralkyl, and the remaining "R" groups not attached to the biomolecule represent hydrogen, a saturated, partially saturated and unsaturated nitrogen-containing cycle or heterocycle. Other preferred groups of compounds are those wherein at least one, but preferably two, of R ₁ or R '1 and R ₂ or R' _2t R 3 or R ' ₃ and R 4 or R' ₄ , R 5 or R ' _s and R e or R'' _e , R7 or R' ₇ and Re or R ' ₈ 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 and unsaturated cyclic radical of 3 to 20 carbon atoms atoms and remaining

9 * 99 9 9 9 * *

99 9 9 9

9 · 9 9,999 9,999 · 9

999999 999

99 * 9 99 ·· 9 9999 The "R groups", in addition to the "R" groups that are attached to the 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 1 or R' _b R ₂ or R ' ₂ and R ₃ or R' _{3 |} R 4 or R ' ₄ and R ₅ or R' _{5 (} Re or R ' _e) , and R ₇ or R' ₇₎ and R e or R ' _a and R 8 or R' ₉ together with the carbon atoms to which they are attached are bonded to 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 a linker are independently selected from hydrogen, saturated, partially saturated and unsaturated cyclic or alkyl groups.

As used herein, "Ř" groups refer to all R groups attached to the carbon atoms of the macrocycle, ie, R, R 1, R ₁ , R _1f, R ₂ , R ' ₂ , Ra, R' ₃ , R 4, R ' ₄ , R _5, R _5, R _6, R _'6), R _7i R _7j Re, Re, R9aR9.

_. Another embodiment of the invention is a pharmaceutical composition in a unit dosage form valid for superoxide dismutation comprising (a) a therapeutically and prophylactically effective amount of a complex as described above, and (b) a non-toxic, pharmaceutically acceptable carrier, adjuvant or vehicle.

A commonly accepted mechanism of action of manganese SOD enzymes involves alternating two oxidation states (II, III) of the manganese center. See J. Bannister, W. H. Bannister, and G. Rotilio, Crit. Roar. Biochem., 22, 111-180 (1987).

1) Μη (II) + HO ₂ ......> Μη (III) + H ₂ O ₂

2) Μπ (III) + O ₂ ......> (η (II) + O ₂

The formal redox potentials of the redox pairs 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 Metaloproteins. Part 1, Metalloproteins with Redox Tasks, ed. Harrison, P. 121. Verlag Chemie (Weinheim, GDR) (1985). For the above mechanism, the potentials of the putative SOD catalyst require the ability to rapidly undergo oxidation state changes in the range of - 0.33 V to 0.87 V.

The complexes derived from Μη (II) and the major class of C-substituted [15] and N ₅ ligands described herein were all characterized by using cyclic voltammetry by measuring their "99".

9 9 9 9 9 · 9 9 9 »9 9 9 9 9»

CT 9 9 9 ·

9 «· and · 9 9 ·, 9 9 9

9 »redox potential. The C-substituted complexes described herein exhibit reversible oxidation at about +0.7 V (SHE). Coulometry has shown that this oxidation is a one-electron process; namely, the oxidation of Μη (II) complex to Μη (III) complex. Therefore, to act as SOD catalysts, the oxidation state Μη (III) is included in the catalytic cycle. That is, the Μη (III) complexes of all these ligands are as suitable as the SOD catalysts, since it does not matter which form ((η (II) or Μη (III)) is present when superoxide is present because superoxide will simply reduce Mn (III) to Μη (II) while releasing oxygen.

As used herein, the term "alkyl", alone or in combination, refers to straight or branched chain alkyl hydrocarbon radicals having 1 to about 22 carbon atoms, preferably from about 1 to 18 carbon atoms, and most preferably from about 1 to about 22 carbon atoms. 12 carbon atoms which optionally carry one or more substituents selected from (1) -NFUnFLi wherein R ™ and _{R 1} are independently selected from hydrogen, alkyl, aryl, or aralkyi; or R ₃₀ is hydrogen, alkyl, aryl or aralkyl, and R ₃₁ is selected from the group consisting of NR 6 R 6 OH 6 OR 6 OR 6

O S O O X '

II-II-II-C-Z; -C-Z '; -S-R35; -S-Rse; -S-R37;

wherein R ₃₂ and R 33 are independently hydrogen, alkyl, aryl or acyl, R 34 is alkyl, aryl or aralkyl, Z 'is hydrogen, alkyl, aryl, alkaryl, -OR 34, -SR 34 or -NR 40 R 41, wherein R 40 and R 41 are independently selected hydrogen, alkyl, aryl or alkaryl, Z 'is alkyl, aryl, alkaryl, -OR 34, -SR 34 or -NR 40 R 41, R 35 is alkyl, aryl, -OR 34 or -NR 40 R 41, R 6 is alkyl, aryl or -NR 40 R 41, R ₃ is alkyl, aryl or alkaryl, X 1 is oxygen or sulfur, and R 3 and R ₃₉ are independently selected from hydrogen, alkyl or aryl; (2) -SR 42, where R 42 is hydrogen, alkyl, aryl, alkaryl, -SR 34, NR 32 R 33,

X 'O o

II II II - C - Z ¹ -, --S - R43, or - ~ P (A) (B);

II

Wherein R 43 is -OH, -OR 34 or-NR 32 R 33, and A and B are independently -OR 34, -SR 34 or -NR 32 R 33;

• 98 9 · *

I (3) - S (= Ο) χ, wherein x is 1 or 2, and R 44 is halide, alkyl, aryl, alkaryl, -OH, -OR 34, -SR 34 or -NR 32 R 33;

(4) -OR 45 wherein R 45 is hydrogen, alkyl, aryl, alkaryl, -NR _{3 2} R 33,

X 'R 44 O 11 II II ^{- c-} Z', ^-s (= O) x, -P-tD) (E), or -P- (R ^ XOR ^);

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

X '

II ...... _: _

- C 4 R 46, (5) wherein R 46 is a halide, -OH, -SH, -OR 34, -SR 34 or -NR 34 R 34; or (6) amine oxides of the formula

AND

With the proviso that R 30 and R 31 are not hydrogen; or

O

II

- P '(F) (G);

(7) wherein F and G are independently -OH, -SH, -OR 34, -SR 34 or -NR 32 R 33; or (8) -O- (- (CH ₂ ) and -O) b -R w, where R 10 is hydrogen or alkyl, and a, b are integers independently selected from 1-6; or (9) halogen, cyano, nitro, or azido. Alkyl, aryl and alkaryl groups on the substituents of the above-defined alkyl groups may contain one substituent for 11, 4, 4, 4 or more, but preferably are unsubstituted. Examples of such hydrocarbon 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, refers to an alkyl hydrocarbon radical having one or more double bonds. Examples of such alkenyl hydrocarbon radicals include, but are not limited to, ethenyl, propenyl, 1-butenyl, cis-2-butenyl, trans-2-butenyl, isobutylenyl, cis-2-pentenyl, trans-2-pentenyl, 3-methyl-1. -butenyl, 2,3-dimethyl-2-butenyl, 1-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, refers to an alkyl hydrocarbon radical having one or more triple bonds. Examples of such alkyne groups include, but are not limited to, ethynylpropinyl (propargyl), 1-butynyl, 1-octynyl, 9-octadecinyl, 1,3-pentadiinyl, 2,4-pentadiinyl, 1,3hexadiinyl, and 2, 4-hexadiinyl. The term "cycloalkyl, alone or in combination, refers to a cycloalkyl hydrocarbon 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 hydrocarbon radicals include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and perhydronaphthyl. The term "cycloalkylalkyl" refers to an alkyl hydrocarbon radical as defined above that is substituted with a cycloalkyl hydrocarbon radical as defined above. Examples of cycloalkylalkyl hydrocarbon radicals include, but are not limited to, cyclohexylmethyl, cyclopentylmethyl, (4isopropylcyclohexyl) methyl, (4-t-butylcyclohexyl) methyl, 3-cyclohexylpropyl, 2cyclohexylmethylpentyl, 3-cyclopentylmethylhexyl, 1- (4-neopentylcyclohexyl), 1- (4-Isopropylcyclohexyl) methylheptyl. The term "cycloalkylcycloalkyl" refers to a cycloalkyl hydrocarbon radical as defined above that is substituted with another cycloalkyl hydrocarbon radical as defined above. Examples of cycloalkylcycloalkyl hydrocarbon radicals include, but are not limited to, cyclohexylcyclopentyl and cyclohexylcyclohexyl. The term "cycloalkenyl", alone or in combination, refers to a cycloalkyl hydrocarbon radical having one or more double bonds. Examples of cycloalkenyl hydrocarbon radicals include, but are not limited to, cyclopentenyl, cyclohexenyl, cyclooctenyl, cyclopentadienyl, cyclohexadienyl, and cyclooctadienyl. The term "cycloalkenylalkyl" refers to an alkyl hydrocarbon radical as defined above which

9 9 9 9

9999 9 is substituted with a cycloalkenyl hydrocarbon radical as defined above. Examples of cycloalkenylalkyl hydrocarbon 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-yl) hexyl, 1- (1-cyclopenten-1-yl) nonyl, and 1 - (1-cyclohexen-1-yl) nonyl. The terms "alkylcycloalkyl" and "alkenylcycloalkyl" refer to a cycloalkyl hydrocarbon radical as defined above which is substituted with an alkyl or alkenyl hydrocarbon radical as defined above. Examples of alkylcycloalkyl and alkenylcycloalkyl hydrocarbon radicals include, but are not limited to, 2-ethylcyclobutyl, 1-methylcyclopentyl, 1-hexylcyclopentyl, 1-methylcyclohexyl, 1- (9-octadecenyl) cyclopentyl and 1- (9-octadecenylphenyl) cyclohexyl. Examples of alkylcycloalkenyl and alkenylcycloalkenyl hydrocarbon radicals include, but are not limited to, 1-methyl-2-cyclopentenyl, 1-hexyl-2cyclopentenyl, 1-ethyl-2, and the like. -cyclohexenyl, 1-butyl-2-cyclohexenyl, 1- (9-octadecenyl) -2cyclohexenyl and 1- (2-pentenyl) -2-cyclohexenyl. The term "aryl", alone or in combination, refers to a phenyl or naphthyl hydrocarbon residue, which optionally carries one or more substituents selected from alkyl, cylcoalkyl, cycloalkenyl, aryl, heterocycles trot, alkoxyaryl, alkaryl, alkoxy, halogen, hydroxy, amine, cyano, nitro, alkylthio, phenoxy, ether, trifluoromethyl 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. The term "aralkyl", alone or in combination, refers to an alkyl or cycloalkyl hydrocarbon radical as defined above wherein one hydrogen atom is replaced with an aryl hydrocarbon radical as defined above, for example, benzyl, 2-phenylethyl, and the like. The term "heterocyclic" refers to ring structures containing in addition to carbon at least one atom of another kind. The most common heteroatoms include nitrogen, oxygen and sulfur. Examples of heterocyclic groups include, but are not limited to, pyrrolidinyl, piperidyl, imidazolidinyl, tetrahydrofuryl, tetrahydrothienyl, furyl, thienyl, pyridyl, quinolyl, isoquinolyl, pyridazinyl, pyrazinyl, indolyl, imidazolyl, oxazolyl, thiazolyl, pyrazolyl, pyridines! triazolyl and tetrazolyl. The term "saturated, partially saturated or unsaturated cyclic" refers to fused cyclic structures in which the 2 carbons of the cycle are also part of a 15-membered macrocyl 13-cyclic ligand. Cyclic structures may contain 3 to 20 carbon atoms, preferably 5 to 10 carbon atoms, and may also contain one or more atoms of another kind in addition to carbon. The most common heteroatoms include nitrogen, oxygen and sulfur. The cyclic structures may also contain more than one cycle. The term "saturated, partially saturated or unsaturated cyclic structure" refers to a cyclic structure in which one carbon of the cycle is also part of a 15 membered macrocyclic ligand. The ring of the structure may contain 3 to 20, preferably 5 to 10, carbon atoms and may also contain nitrogen, oxygen and / or sulfur atoms. The term "nitrogen-containing heterocycle" refers to cyclic structures in which 2 carbons and the nitrogen of the cycle are also part of a 15-membered macrocyclic ligand. The cyclic structure may contain 2 to 20, preferably 4 to 10, carbon atoms, may be partially or fully unsaturated or saturated, and may also contain nitrogen, oxygen and / or sulfur atoms in the ring portion, which is not also part of a 15 membered macrocyclic ligand. The term "anion-organic acids" refers to carboxylic acid anions of about 1 to about 18 carbon atoms. The term " helogenidium "

The macrocyclic ligands useful in the complexes of the invention can be prepared according to the general procedure shown in Scheme A explained below. Thus, the amino acid amide, which is the corresponding amido derivative of a naturally occurring or non-naturally occurring α-amino acid, is limited to the form of the corresponding substituted ethylenediamine. Such an amino acid amide may be an amido derivative of any of a number of well known amino acids. Preferred amino acid amides are represented by the formula:

H where R is derived from the D or L forms of 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 non-naturally occurring α-amino acids, such as alkyl, ethyl, butyl, tert-butyl, cycloalkyl, phenyl, alkenyl, allyl, alkynyl, aryl, heteroaryl, polycycloalkyl, polycycloaryl, polycycloheteroaryl, imines , aminoalkyl, hydroxyalkyl, # · alkyl · # # # amino amino amino amino amino amino amino amino amino amino amino amino amino amino amino amino amino amino amino * Yl 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, and midy, amino acids, peptides, proteins, carbohydrates, nucleic acids, fatty acids, lipids, nitro, hydroxylamines, hydroxamic acids, thiocarbonyls, borates, boranes, borases, silyl, siloxy, silase, and combinations thereof. Most preferred are those wherein R represents hydrogen, alkyl, cycloalkylalkyl, and aralkyl. The diamine is then tosylated to form the di-N-tosylated derivative, which reacts with the di-O-tosylated tris-N-tosylated triazaalkanediol to give the corresponding substituted N-pentatosylpentaazacycloalkane. The tosyl groups are then removed and the resulting compound reacts with the manganese (II) compound under necessarily anhydrous and anaerobic conditions to form the corresponding substituted manganese (II) pentaazacycloalkane complex. If the charge neutralizing or anionic ligands, for example X, Y 2, are anions or ligands that cannot be introduced directly from the manganese compound, the complex with these anions or the tiger can be formed by carrying out an exchange reaction with the complex prepared by reacting the macrocycle with the manganese compound.

Complexes according to the invention, wherein R ₉ and R ₂ is alkyl, and R 3, R ' ₃ ' R 4, R ' ₄ , R 5, R', R ', R' e, R 7, R ' ₇ , R e and R' e may be alkyl, arylalkyl or cycloalkylalkyl and R or R 'and R 1 or R' 1 together with carbon atoms, to which they are attached, are attached to form a nitrogen-containing heterocycle, may also be prepared according to the general procedure shown in Scheme B below using techniques known to those skilled in the art to prepare a precursor of pentaazabicyclo [12.3.1] manganese (II) octadecapentaene (II) complex . See, for example, Alexander et al., Inorg. Nucl. Chem. Lett., 6,445 (1970). Thus, 2,6-diketopyridine is condensed with triethylenetetraamine in the presence of a manganese (II) compound to form a pentaazabicyclo [12.3.1] manganese (II) ioctadecapentaene (II) complex; platinum (10-1000 psi) to give the corresponding pentaazabicyclo (12,3,1) octadecatriene manganese (II) complex.

The macrocyclic ligands useful in the complexes of the invention may be prepared via the dibasic acid dichloride shown in Scheme C below. Thus, the triazaalkane is tosylated in a suitable solvent system to form the corresponding tris (N-tosyl) derivative. Such a derivative is treated with a suitable base to form the corresponding disulfonamide anion. The disulfonamide anion is dialkylated by a suitable one

4 * * • 3 ·

4 4 ♦ 443 4>

44 • · · · 4 4 · * 4 4

444 * 44 * 4 * 4 * 4 * * by an electrophile to form a dicarboxylic acid derivative. This dicarboxylic acid derivative is converted to a dicarboxylic acid, which is then treated with a suitable reagent to form the dibasic acid dichloride. The desired vicinal diamine is obtained by any of several routes. One route that is useful is the preparation of an aldehyde by reaction with cyanide in the presence of ammonium chloride followed by treatment with acid to form α-ammonium nitrile. The latter compound is reduced in the presence of an acid and then treated with a suitable base to form a vicinal diamine. Condensation of the dibasic acid dichloride with the vicinal diamine in the presence of a suitable base gives rise to the macrocycle tris (tolyl) diamide. The tosyl groups are removed and the amides are reduced, and the resulting compound is reacted with the manganese (II) compound under substantially anhydrous and anaerobic conditions to form the corresponding substituted pentaazacycycloalkane (II) complex.

Vicinal diamines were prepared by the route shown (known as Strecker synthesis) and vicinal diamines were purchased if commercially available. Any method of preparing a vicinal diamine could be used.

The macrocyclic ligands useful in the complexes of the invention may also be prepared by the pyridine diamide route shown in Scheme D below. Thus, a polyamine such as a tetraaza compound containing two primary amines is condensed with dimethyl 2,6-pyridinedicarboxylate by heating in a suitable solvent, for example methanol, to form a macrocycle incorporating a pyridine cycle, for example 2,6-dicarboxamide. The pyridine cycle in the macrocycle is reduced to the corresponding piperidine cycle in the macrocycle, and then these diamides are reduced and the resulting compound reacts with the manganese (II) compound under substantially anhydrous and anaerobic conditions to form the corresponding pentaazacyclloalkane (II) complex.

The macrocyclic ligands useful in the complexes of the invention may also be prepared by the bis (haloacetamide) route shown in Scheme E below. Thus, the triazaalkane is tosylated in a suitable solvent system to form the corresponding tris (N-tosyl) derivative. Such a derivative is treated with a suitable base to form the corresponding disulfonamide anion. A bis (haloacetamide), for example bis (chloroacetamide), of a vicinal diamine is prepared by reacting the diamine with an excess of a haloacetyl halide, for example chloroacetyl chloride, in the presence of a base. The tris (N-tosyl) triazaalkane disulfonamide anion then reacts with the bis (chloroacetamide) diamine to form a substituted tris (N-tosyl) diamide macrocycle. The tosyl groups are eliminated w

9

9 · · ·

9999 98 99

9

9 9

9999 ·

9 9

The nene and amides are reduced and the resulting compound reacts with the manganese (II) compound under substantially anhydrous and anaerobic conditions to form the corresponding manganese (II) pentaazacycloalkane (II) complex.

The macrocyclic ligands useful in the complexes of the invention wherein R _1f R _{1 (R 2 l} R x are derived from the starting diamino compounds and R _5, R _{'5 l} R _7, R' ₇₎ and R _9, R ₉ can be H or a functional group described previously, can be prepared according to the pseudopeptide method shown in Scheme F below. Substituted 1,2-diaminoethane represented by the formula

H ₂ N nh ₂ wherein R _{1 (} R 1, R ₂ and R ' ₂ are substituents on adjacent carbon atoms in the result of a small cyclic cyclic ligand as described above may be used in this method in combination with any amino acids. The diamine may be produced by any conventional method known to those skilled in the art. given area. R groups in the macrocycle derived from substituents on the a-carbon of α-amino acids, e.g., R _{5 l} R _'5, R _7, R' _7, R ₉ and R _'9 can be derived from 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, lilac, tryptophan, threonine, tyrosine, valine and / or R groups of non-natural α-amino acids alkyl, ethyl, butyl, tert-butyl, cycloalkyl, phenyl, alkenyl, allyl, alkynyl, aryl, heteroaryl, polycycloalkyl, polycycloaryl, polycycloheteroaryl, imine, aminoalkyl, hydroxyalkyl, hydroxyl yl, phenol, amine oxides, thioalkyl, carboalkoxyalkyl, carboxylic acids and derivatives thereof, keto, ether, aldehyde, amine, nitrile, halo, thiol, sulfoxide, sulfone, sulfonic acids, sulfide, disulfide, phosphonic acids, phosphinic acids, phosphinexides, sulfonamides , amides, amino acids, peptides, proteins, carbohydrates, nucleic acids, fatty acids, lipids, nitro, hydroxylamines, hydroxamic acids, thiocarbonyl, borates, boranes, borase, silyl, siloxy, silaza, and combinations thereof. For example, 1,8-dihydroxy-4,5-diaminoctane is monotosylated and reacted with Boc anhydride to provide a differentiated N-Boc, N-tosyl derivative. The sulfonamide was alkylated with methyl bromoacetate using sodium hydride as base and saponified to the free acid. Diamine containing * 9

99,999 • 9 9 9 9 9 *

9 9 · 9 9 9 9 999 • 9 «E * 99999 * 9999 9 • 99999 999 ••« * 99 99 9 ·· · *

N-tosylglycine serves as a dipeptide substitute for standard aqueous phase peptide synthesis. Thus, a corresponding pseudo-tripeptide is obtained by coupling with an amino acid ester with different functional groups. The two subsequent TFA cleavage and binding results in a pseudo-pentapeptide which can be deprotected at the N- and C-termini in one step using HCl / AcOH. DPPA-mediated cyclization followed by reduction of L1AIH4 or borane provides the corresponding macrocyclic ligand. This ligand system reacts with a manganese (II) compound, for example with manganese chloride, under essentially anaerobic conditions to form the corresponding functionalized manganese (II) pentaazacycycloalkane (II) complex. When the ligands or charge neutralizing anions, for example X, Y and Z, are anions or ligands that cannot be introduced directly from the manganese compound, a complex with these anions or ligands can be formed by performing an exchange reaction with a complex prepared by the macrocycle reaction with the LQ compound. of manganese. ——— -— --—. The macrocyclic ligands useful in the complexes of the invention wherein R 1, R 1, R 3, R 3, R 3, R 3, R 3 R 3, R 3, R 3, R 3, or R 3 may be H or any of the functional groups described above can be prepared according to the general peptide method shown in Scheme G below. The R groups in the macrocycle derived from the substituents on the α-carbon of amino acids, for example R _{1 (} R _1b R 3, R ' ₃₎ R ₅ , R's, R 7, R 8, R 8 and R' ₉ are defined above in Scheme F. the cyclic peptide precursors from the corresponding linear peptides are the same or significant modifications to methods known in the art. See, for example, Veber, DF et al., J. Org. Chem., 44, 3101 (1979). The general method outlined in Scheme G below is an example using sequential preparation of a functionalized linear pentapeptide from the N-terminus or C-terminus in the liquid phase. Alternatively, the reaction sequence for the preparation of the linear pentapeptide may be carried out using solid phase methods known in the art. The reaction sequence can be performed from the C-terminus to the N-terminus and by different approaches such as by coupling di- and tri-peptides as needed. Thus, the Boc-protected amino acid is coupled to an amino acid ester using standard peptide coupling reagents. The new Bocdipeptide ester is then saponified to the free acid, which is again attached to another amino acid ester. The resulting Boc-tripeptide ester is saponified and the method continues until the Boc-protected pentapeptide free acid is prepared. The Boc protecting group is removed under standard conditions and the resulting pentapeptide or salt thereof is converted to a cyclic pentapeptide. The cyclic pentapeptide is then reduced to pentaazacyclopentadecane with lithium aluminum hydride or borane. The final ligand then reacts with the manganese (II) compound under essentially anaerobic conditions to form the corresponding pentaazacyclopentadecane manganese (II) complex. If the ligands or charge-neutralizing anions such as X, Y and Z are anions or ligands that cannot be introduced directly from the manganese compound, the complex with these anions or ligands can be formed by performing an exchange reaction with the complex prepared by the macrocycle reaction with a manganese compound.

Scheme B

AAA · ·

A AA A AAA

A A ·

AAAA AA AA

A A A A A AAA

AAAA ·

A A A

A A AA

9 «

9 9 ·

9 99

98 «9 9

9 9 «

Scheme A

9 «*« ·

9 «

9 9

9999 ♦>

9 * 9 9 9

9 9 9 • 9 9

9 «

9

LiAW / THF

T «HN

TsQ / EijN

CH 3 Cl 2

* · A A A A A

* AAA

AAA AAAA •• AA AA AA A

A A 4

A A A

AAAA 4

A A 4

A A A A

Scheme C

A * O

ι.τ »α, 'Na ·. pyrttfn _T «-N *

N <

1. KCN. NHLO ^ QKlhf

2.NaOC ₂ H c ₂ h ₉ oh ⁵ R.

Na * Ts

BrCR ₂ COOCH ₃ (trf-Cl

Scheme D • 9 99 9 »9

9 9 9 9 * * · ··

9 «9 9999

9 9 9 «<

0999 99 99

9 9 9

9 9 9

9 9 9

9

9 9

99

H ₂ 7PtO ₂ - CH ₃ OH HQ

9 9 9 i 9 9 4

I

9 * 4

9 4

99

9 9 9 zo

9 «• 9 I I» (• 99999

Scheme F • · ·

9

9

9 9 9

9999 99 99 • 9 • A

• 9

9

I «* 9 ♦ 999 8 ·

99 9 9 9 9 9 9

9 9

99

Diagram F (continued)

9

Scheme G fy n '.

4 «99

9 9 9

9 ·

9 «

9 *

9999 8 · ·

999 9 • 9 9

9 9

9 9

99 9 9

9 ·

99

NaOH, H 2 O CH 3 OH

EDC-HCl. HOBT, OMF.TEA.RT ethyl chloroformate OMF, TEA, 0 ° C

BocM

EDC-HCl. Hobt DMF, TEA. RT 'or ethyl chloroformate dmf.tea, ° C

—Edc-hci, hobt. DMF.TEA.RT or DMF ethyl chloroformate, TEA, o * c

NaOH, H 2 O CH 3 OH

EDC-HCl, HOBT DMF. TEA. RT or ethyl chloroform-dmf, thioc 9

9 9 • 9 * 99

9 * 9 9

9

9 · 9 9

9999 9 * 9 9

9 4

9 9 * 9 I »a

9 9 9

Diagram G (continued)

/ uah.

/ THF. δ f or

BHj. THF

4 4

4 4 *

4 · 4444 4 444

4

• 4 · «44

The pentaazamacrocycles of the invention may have one or more carbon atoms and are therefore capable of existing in the form of optical isomers as well as in the form of their racemic or non-racemic mixtures. Optical isomers may be obtained by resolution of the racemic mixtures according to conventional procedures, for example by formation of diastereoisomeric salts by treatment with an optically active acid. Examples of suitable acids are tartaric, diacetyltartaric, dibenzoyltartaric, ditoluoyltartaric, and camphorsulfonic acid and then separation of the mixture of diastereoisomers by crystallization followed by liberation of optically active bases from these salts. A different procedure for separation of optical isomers involves the use of a chiral chromatography column optimally selected to maximize separation of enantiomers. Yet another useful method involves the synthesis of covalent diastereoisomeric molecules by reacting one or more secondary amino groups of the compounds of the invention with an optically pure acid in activated form or an optically pure isocyanate. The synthesized diastereoisomers may be separated by conventional means such as chromatography, distillation, crystallization or sublimation, and then hydrolyzed to enantiomerically ^- pure ligands to obtain. The optically active compounds of the invention may also be obtained using optically active starting materials, such as amino acids.

The compounds and complexes of the invention are novel and can be used to treat numerous inflammatory disease states and disorders. For example, reperfusion injury to the ischemic organ, for example, reperfusion injury to the ischemic myocardium, surgically induced ischemia, inflammatory disease of the internal organs, rheumatoid arthritis, osteoarthritis, psoriasis, organ transplant rejection, radiation-induced damage, oxidatively-induced damage and tissue destruction, thrombosis, stroke, acute pancreatitis, insulin-dependent diabetes mellitus, widespread intravascular coagulation, fat embolism, adult and infantile respiratory stress, metastasis and carcinogenesis.

The activity of the compounds or complexes of the invention for catalysing superoxide dismutation can be demonstrated using a stopped flow kinetic analysis as described in Riley, D.P., Rivers, W.J. and Weiss, R.H., "Stop Flow Flow Kinetic Analysis for Monitoring Superoxide Decomposition in Water Systems", Anal. Biochem., 196, 344-349 (1991), which is incorporated herein by reference. Stopped flow kinetic analysis is an accurate and direct method for quantitative monitoring of superoxide degradation rates in water. Steady-state kinetic analysis is suitable for screening compounds for SOD activity and catalytic activity of SODs.

The compounds or compositions of the invention for superoxide dismutation as shown by the arrest flow analysis are related to the treatment of the aforementioned disease states and disorders.

The total daily dose administered to the host in single or divided doses may be in amounts, for example, of about 1 to 100 mg / kg body weight per day, and more usually about 3 to 30 mg / kg. Unit dose compositions may contain such amounts of their proportions that they constitute a daily dose.

The amount of active ingredient that may be combined with the carrier materials to form a single dosage form will vary depending upon the host treated and the particular mode of administration.

The dosage diet for the treatment of disease states with the compounds and / or compositions of the invention is selected in accordance with a variety of factors including type, age, weight, sex, medical condition of the patient, severity of the disease, route of administration, pharmacological considerations and efficacy.Efficiency, pharmacokinetictoxicologicprofilesconcrete used; Thus, the currently used dosing diet can vary widely and thus can deviate from the preferred dosing diet listed above.

The compounds of the invention may be administered orally, parenterally, by inhalation spray, rectally, or topically in unit dosage forms containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired. Topical administration may include the use of transdermal administration, 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. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-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

9

AA AA • AA · AA AA AA AA «« «AA«

AAAAAA · ·

Synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid have found utility in the preparation of injectables.

Suppositories for rectal administration of the drug may be prepared by mixing the drug with a suitable non-irritating excipient such as cocoa butter and polyethylene glycols that are solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt and release the drug.

Solid dosage forms for oral administration may include capsules, tablets, pills, powders, granules, and gels. In such solid dosage forms, the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such a dosage form may also include, as in conventional practice, additives other than inert diluents, for example, lubricating agents such as magnesium stearate. In the case of capsules, tablets and pills, the dosage forms may also contain buffering agents. Tablets and pills may additionally be prepared 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 contain adjuvants such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring, and fragrant agents.

While the compounds of the invention may be administered as a separate active pharmaceutical agent, they may also be used in combination with one or more compounds which are known to have an effect against a particular disease state to which treatment is directed.

The compounds or complexes of the invention may also be used as MRI contrast agents. A discussion of the use of contrast agents in MRI can be found in U.S. Patent Application Serial No. 08 / 397,469, which is incorporated herein by reference.

The contemplated equivalents of the formulas above for compounds and derivatives as well as for intermediates are compounds corresponding thereto and with the same general properties as the tautomers of the compounds and wherein, for example, one or more different R groups are simple substituent variations as defined herein, for example, wherein R is a higher alkyl group than the latter, or wherein the tosyl groups are different nitrogen or oxygen protecting groups, or wherein O-tosyl is a halide. Anions with a charge other than 1, such as carbonate, phosphate and dibasic phosphate, may be used in place of anions with a charge of 1 if they do not adversely affect the overall activity of the complex. However, the use of anions with a charge other than 1 will result in a slight modification of the general formula (I) for the complex mentioned above. In addition, where a substituent is designated as, or may be, hydrogen, the exact chemical nature of a substituent other than hydrogen at this position, such as a hydrocarbon residue or halogen, hydroxy, amino and the like, is not critical as long as it does not adversely affect overall activity and / or the synthesis process. It is further contemplated that the manganese (III) complexes will be equivalent to the subject manganese (II) complexes.

The chemical reactions described above are generally known in terms of their broadest application for the preparation of the compounds of the invention. Occasionally, the reactions may not be applicable as described for each compound contained within the scope of the disclosure. Compounds to which this applies will be readily recognized by those skilled in the art. In all these cases. the reactions may also be successfully performed by conventional modifications known to those skilled in the art, for example, by suitable protection of the interfering groups with exchange and / or alternative reagents, routine modification of reaction conditions, and the like, or by other reactions known in the art or otherwise conventional methods will be useful for preparing the corresponding compounds of the invention. In all preparation procedures, all starting materials are known or can be easily prepared from known starting materials.

Without further elaboration, it is believed that one skilled in the art can, using the preceding descriptions, utilize the invention to its fullest extent. The following preferred specific embodiments should, therefore, be construed as merely illustrative and non-limiting of the remainder of the specification.

DETAILED DESCRIPTION OF THE INVENTION

All reagents were used as obtained 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 performed on Finigan MAT90, Finigan 4500 and VG40-250T using m-nitrobenzyl alcohol (NBA), m-nitrobenzyl alcohol / LiCl (NBA - Li). Melting points are uncorrected.

9 9 9

9 9 • ·

4

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

Ala L-alanine DAla D-alanine Gly glycine Ser serine DSer D-serine Bzl benzyl Boc tert-butoxycarbonyl Et ethyl TFA trifluoroacetic acid DMF dimethylformamide HOBTÍH ₂ O 1-hydroxy- (1H) -benzotriazole monohydrate EDOHCI 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride TEA triethylamine DMSO dimethylsulfoxide THF tetrahydrofuran DPPA diphenylphosphoryl azide

Yc Cyc stands for 1,2-cyclohexanediamine (stereochemistry, for example R, R or S, S is indicated as such). This allows the three-letter peptide nomenclature code to be used for pseudopeptides containing a 1,2-cyclohexanediamine "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 mol) in CH ₂ Cl ₂ (5.00 L) at -10 ° C was added a solution of p-toluenesulfonyl chloride (209 g, 1 L). , 10 mol) in CH ₂ Cl ₂ (5.00 L) dropwise over a 7 h period, maintaining a temperature of -5 ° C to -10 ° C. The mixture was allowed to warm to room temperature while stirring overnight. 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 (10 x 1 L) and dried over MgSO ₄ . Removal of solvent in • 9 99 * 999 ·· «9 9 9 9

999999 · 999 99 99 * 9 · «9 vacuum gave 286 g (97.5% yield) of the product as a yellow crystalline solid: ¹ H NMR (CDCl 3) δ 0.98-1.27 (m, 4 H) 1.54 -1.66 (m, 2H), 1.81-1.93 (m, 2H), 2.34 (dt, J = 4.0, 10.7 Hz, 1H), 2.42 (s, 3H), 2.62 (dt, J = 4.2, 9.9 Hz, 1H), 7.29 (d, J = 8.1 Hz, 2H), 7, 77 (d, J = 8.3 Hz, 2H); MS (LRFAB-DTT-DTE) m / z 269 [M + H] &lt; + &gt; ^.

B. Synthesis of N- (p-toluenesulfonyl) -N '- (Boc) - (R, R) -1,2-diamino-cyclohexane

To a stirred solution of N- (p-toluenesulfonyl) - (R, R) -1,2-diaminocyclohexane prepared as in Example 1A (265 g, 0.955 mol) in THF (1.15 L) was added 1 M aqueous NaOH ( 1.15 L, 1.15 mol). Di-t-butyl dicarbonate (229 g, 1.05 mol) was then added and the resulting mixture was stirred overnight. The layers were separated and the aqueous layer was adjusted to pH 2 with 1 M HCl and saturated with NaCl. The aqueous solution was then extracted with CH ₂ Cl ₂ (2 x 500 mL) and the extracts and 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 THF-ether-hexanes to give 310 g (88.1% yield) of the product as a white crystalline solid: mp 137-139 ° 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 (brs, 1H), 3.30 (br d, J = 9.6 Hz, 1H), 4, 37 (br d, J = 6.7 Hz, 1H) 5.48 (br d, J = 4.6 Hz, 1H), 7.27 (d, J = 9.7 Hz, 2H), 7.73 (d, J = 8.1 Hz, 2H); MS (LRFAB, NBA Li) m / z 375 [M + Lif.

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

To a stirred solution of N- (p-toluenesulfonyl) -N '- (Boc) - (R, R) -1,2-diamino cyclohexane prepared as in Example 1B (310 g, 0.841 mol) in anhydrous DMF (3.11 L) NaH (37.4 g - 60% in oil, 0.934 mol) was added portionwise at 0 ° C and the resulting mixture was stirred for 30 minutes. Methyl bromoacetate (142 g, 0.925 mol) was then added dropwise over 45 minutes and the mixture was allowed to warm to room temperature while 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 was dried over MgSO ₄ . The solvent was removed in vacuo and the resulting oil was dissolved in ether. Crystallization by addition of hexanes gave 364 g (98% yield) of the product (TLC (98: 2) CHCl ₃ MeOH / silica gel / UV detection) showed the product contained about 5% starting material) as colorless needles: -2 ° C; ¹ H NMR

9 4 · * 44

445 4 4 4 · «445

4 4 4 «4 4 949 4 4 ** 4 * * 4 ** ·· 449 • 444 44 44 4 44 44 (CDCl 3) 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 2 H), 3.68 (s, 3 H), 4.06 (ABq, J = 18.5 Hz, J = 155 Hz, 2 H), 4.77 (br s, 1H), 7, 30 (d, J = 8.3 Hz, 2H), 7.82 (d, J = 8.3 Hz, 2H); MS (LRFAB, DTT-DTE) m / z 441 [M + H] +

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

To a stirred solution of the impure Boc- (R, R) -Cyc (Ts) -Gly-OMe prepared as in Example 1C (217 g, 0.492 mol) in MeOH (1.05 L) was slowly added a 2.5 M aqueous solution of 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 water (1.5 L). The solution was filtered to remove a small amount of solid and washed with ether (7 X 1 L) to remove the impurity (compound 1B) which, after drying the combined washes over MgSO ₄ and removing the solvent in vacuo, yielded 8.37 g. The aqueous solution was then adjusted to pH 2 with 1 M 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 remaining 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, 9H), 1.55 -1.68 (m, 3H), 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, λυ = 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, 2H), 8.81 (br s, 1H); MS (LRFAB, NBA-Li) mlz 433 [M + Li] ⁺ .

E. Synthesis of Boc- (R, R) -Cyc (Ts) -Gly-Gly-Gly-OEt

To Boc- (R, R) -Cyc (Ts) -Gly-OH (18.1 g, 43.1 mmof) in DMF (480 mL) was added HOBt <H ₂ O (7.92 g, 51.7 mmol) and EDOHCl (9.91 g, 51.7 mmol) and the resulting mixture was stirred for 20 min at room temperature. 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 then stirred for 16 hours. 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 1 M 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, 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, 1H), 2 , 38 (s, 3H), 3.30-3.40 (bs, 2H), 3.75 35 • * • ♦♦

4 4 4

4 ·· «4445 4

4 4

44 44

4.01 (complex m, 4 H), 4.08 (q, J = 7.6 Hz, 2H), 6.05 (bs, 1H), 7.32 (d, J = 8.0 Hz) 2 H), 7.77 (d, J = 8.0 Hz, 2H), 8.32 (t, J = 7.2 Hz, 1H); MS (HRFAB) m / z 518.2551 (M + H +; 518.2512 calcd for C 24 H 37 N 3 O 7 SSi).

F. Synthesis 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 CH ₂ Cl ₂ (180 mL) was added TFA (44 mL) and the resulting mixture was stirred at room temperature for for 30 minutes. The solution was concentrated and the residue was dissolved in ether (50 mL) and precipitated with hexane (500 mL). The solvents were decanted and the residue was washed with 10: 1 hexane / ether (500 mL). The final residue was thoroughly dried under high vacuum to give 20.7 g (95% yield) of the product as a tan foam: ¹ H NMR (DMSOde) δ 0.85 - 0.96 (m, 1 H), 1.03 - 1.31 (complex m, 7 H), 1.09 (t, J = 7.6 Hz, 3H), 2.00. (m, H _1) _ 2,39_ (s _3_H) _l _3. 02_ (bs, J_H) _3,62 _L ^ (m, H), - 3.82 --- 4 ₇ 05- _(m-r 4 H) _{r r} 4 10- (qH- = 7.6 Hz, 2 H), 7.41 (d, J = 8.0 Hz, 2H), 7.67 (d, J = 8.0 Hz, 2H), 8.25 (bs, 3 H), 9.09 (t, J = 5; 637rH) rMS (HRFABpn / z 418.1990 (Μ -ΎΎ + Τϊ) Τ418.1988 calcd for C 18 H 14 NaO 5 S).

G. Synthesis of Boc-Orn (Z) -Cyc (Ts) -Gly-Gly-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 EDOHCl (5.25) g, 27.4 mmol) and the resulting solution was stirred for 20 minutes at room temperature. To this solution was added Cyc (Ts) -Gly-GlyOEt TFA salt (12.0 g, 22.8 mmol) and TEA (3.82 mL, 27.4 mmol) and stirring was continued for 16 hours. 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 1 M 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-d _e) δ 1.00 to 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, 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), 7.20-7.37 (m, 8H), 7.78 (m, 2H), 8.30 (bs, 1 H); MS (LRFAB, NBA + HCl) m / z 760 (M + H +).

A A AA A A A A A A

AAAA AA

AA · ♦ *

A A AA

A A A A A A A A A A A

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

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 solution was Stir at room temperature for 30 minutes. The solution was evaporated and the residue was triturated with ether (100 mL). The ether was decanted and the residue was thoroughly dried under high vacuum to give 15.5 g (> 100% yield, containing TFA) of the product as an orange foam: ¹ K NMR (DMSO-d ₆ ) δ 0.97 L, 93 (complex 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, 2H), 3.71-3.91 (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 (bs, 3H), 8 .41 (bs, 1H); MS (LRFAB, NBA + HCl) m / z 660 (M-TFA) ^&lt; ^{+ &gt;} .

I. Synthesis of Boc-Gly-Orn (Z) -Cyc (Ts) -Gly-Gly-OEt - To a solution of Boc-Gly-OH (3.36 g, 19.2 mmol) in DMF (2320 mL) was added HOBt H ₂ O (3.52 g, 23.0 mmol) and EDOHCl (4.41 g, 23 _with 0 mmol) and the resulting solution was stirred for 20 minutes at room temperature. 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 continued for for 12 hours. 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 1 M KHSO 4 (150 mL), water (150 mL), saturated NaHCO ₃ (150 mL) and brine (150 mL) and dried (MgSCM. Filtration and concentration gave 13.7 g ( 87% yield) of product as a white foam: 'H NMR (DMSO-d _e) δ 0.96 -1.10 (m, 2 H), 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 (bs, 2H), 3.67 3 84 (m, 4H), 3.93-4.03 (m, 3H), 4.06 (q, J = 7.4 Hz, 2H), 6.92 (bs, 1H), 7.19 (m, 1H), 7.24-7.37 (m, 7H), 7.60 (d, J = 8.3 Hz, 1H), 7.76 (m, 2H) 7.38 (bs, 1H) MS (LRFAB, NBA + Li) ⁺ m / z 823 (M + Li) ⁺ .

J. Synthesis of Boc-Gly-Orn (Z) -Cyc (Ts) -Gly-Gly-OH

To a solution of Boc-Gly-Orn (Z) -Cyc (Ts) -Gly-Gly-OEt (13.3 g, 16.3 mmol) in methanol (100 mL) was added 1 M NaOH (25 mL). The resulting mixture was stirred at room temperature and monitored by TLC. After 2 hours the reaction was complete. 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 removed. The pH was lowered to 3.5 with 1 M KHSO 4 and the aqueous phase was extracted with EtOAc (3 x 100 mL). The combined EtOAc layers were dried (MgSO ₄ ), »

* «

4 4 · 4 4

4 4 4 44444 «

4 4 4 4

4444 44 44 filtr filtered and concentrated to give 11.7 g (91% yield) of the product as a white foam: 1 H NMR (CDCl 3) δ 0.98 -1.25 (m, 2H), 1.38 (s, 9H), 1.40-1.92 (m, 10H), 2.38 (s, 3H), 2.97 (m, 2H), 3.62 (bs, 2H), 3, 75-3.85 (m, 3H), 3.95-4.05 (m, 2H), 5.01 (s, 2H), 6.96 (bs, 1H), 7.28 ( m, 1H), 7.25-7.38 (m, 7H), 7.61 (d, J = 8.4 Hz, 1H), 7.78 (m, 2H), 8.25 (bs, 1H).

K. Synthesis of Gly-Orn (Z) -Cyc (Ts) -Gly-Gly-OH TFA salts

To a solution of Boc-Gly-Orn (Z) -Cyc (Ts) -Gly-Gly-OH (11.2 g, 14.3 mmol) in CH ₂ Cl ₂ (100 mL) was added TFA (24 mL) and the resulting solution was stirred for 30 minutes at room temperature. The solution was concentrated and triturated with ethyl ether (500 mL). Filtration gave 11.3 g (99% yield) of the product as a white powder: ¹ H NMR (DMSOde) δ 0.95 -1.98 (complex m, 12 H), 2.39 (s, 3 H), 3 1.01 (m, 2H), 3.38 (m, 1H), 3.65 4.10 (complex m, 7H), 4.18 (q, J = 2.4Hz, JLH). .Delta., 2.02_ (s, 2H), -7.24, -7, -40- (m, -9-H-) _r 7.77-7.85 (m, 2H), 8.13 (bs, 3H), 8.31 (bs, 1H), 8.42 (d, J = 8.3 Hz, 1H); MS (HRFAB) 689: 2953 (M-TFA) ⁺ ; ^_ 689; 2969 výpoČtěhopřo'C ^_ ^ ^ H ~ N609S "

L. Synthesis of cyWo- (Gly-Orn (Z) -Cyc (Ts) -Gly-Gly-)

A solution of Gly-Orn (Z) -Cyc (Ts) -Gly-Gly-OH TFA salt (5.0 g, 6.23 mmol) in dry degassed DMF (1520 mL) was treated with TEA (1.74 mL, 12 mL), (5 mmol) and the solution was cooled to -40 ° C. DPPA (1.64 mL, 7.60 mmol) was added dropwise over 10 minutes and the reaction was then stirred at -40 ° C for 3 h. After this time, the reaction was placed in a -2 ° C bath. and then allowed to stand at this temperature for 16 h. Water (1520 mL) was added and the resulting solution was stirred with a mixed ion exchanger cartridge (750 g) for 6 hours at room temperature. The resin was then filtered and the solution was concentrated to a volume of about 100 mL (DMF). Upon addition of ethyl ether (500 mL), a solid residue formed which was redissolved in methanol (100 mL) and reprecipitated by addition of ethyl ether (500 mL). Filtration gave 3.26 g (78% yield) of the product as a white powder: ¹ H NMR (CDCl ₃ ) δ 0.96 - 2.10 (complex m, 14 H), 2.37 (bs, 3 H), 2.68-3.05 (m, 3H), 3.42-3.90 (complex m, 8H), 4.1.4 (m, 1H), 4.20 (m, 1H), 4 , 97 5.08 (m, 3H), 6.42 (d, J = 8.4 Hz, 1H), 7.19 (d, J = 8.0 Hz, 1H), 7.20 - 7.39 (m, 7H), 7.65-7.78 (m, 2H), 9.15 (bs, 1H), 9.22 (bs, 1H); MS (HRFAB) m / z 671.2842 (M + H) ^&lt; ^{+ &gt};; 671.2863 calcd for C 14 H 14 N 5 O 5 S.

φ »« 9 99

ΦΦΦΦ φ · · φ 9 9 9 9 9 9 9 9 • • • •

«99 9 9 9 999

Μ. Synthesis of cyWo- (Gly-Om-Cyc (Ts) -Gly-Gly-)

Κ a solution of cyWo- (Gly-Om (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 heated at reflux for 2 hours and then 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-d _e) δ 0.94 to 2.22 (complex m, 12 H), 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, 1H), 7.27 (d, J = 9.3 Hz, 1H), 7.25- 7.45 (m, 2H), 7.64-7.80 (m, 2H), 9.12-9.29 (m, 2H); MS (HRFAB) m / z 537.2511 (M + H) ^&lt; ^{+ &gt};; 537.2495 calcd.

N. cy / r / o- (Gly-Om (lithocholyl) -Cyc (Ts) -Gly-Gly-) Synthesis

To a solution of cyWo- (Gly-Om-Cyc (Ts) -Gly-Gly-) (1.0 g, 1.9 mmol) in CHCl 3 (25 mL) was added NHS-active ester of lithocholic acid (881 mg, 1.9 mmol) and the resulting mixture was then stirred for 16 hours after addition of ethyl ether (50 mL) to form a solid. Filtration gave 946 mg (56% yield) of the product as a tan powder: ¹ H NMR (CD 3 OD) δ 0.66 (m, 3 H), 0.93 (bs, 6 h), 0.94-2.37 ( complex m, 48 H), 2.43 (s, 3 H), 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) ^&lt; ^{+ &gt};; 895.5367 calculated for C ₄ H ₇ N ₆ O ₈ S.

O. Synthesis of 2,3- (R, R) -cyclohexano-6- (S) - {3- (lithochoiylamino) propyl} -1,4,7,10,13pentaazacyclopentadecane

To a suspension of cy / r / o- (Gly-Om (lithocholyl) -Cyc (Ts) -Gly-Gly-) (2.70 g, 3.00 mmol) in THF (50 mL) was added lithium aluminum hydride (51, 0 ml of 1.0 M solution). The resulting mixture was then heated to reflux for 16 hours. The reaction mixture was cooled to about -20 ° C and quenched (cautiously) with 5% Na ₂ SO ₄ (30 mL) followed by methanol (30 mL). This solution was stirred at room temperature for 1 hour and concentrated to 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 give 800 mg (40% yield) of the product as a colorless oil: ¹ H NMR (C ⁶ D 6) δ 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, 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;

98 99 9> 9 9 9 · 9 9

99 · 999

9 9 9 9 9 99

9 · 99 9 • 999 99> 9 9 • 9 99

9 • • 99

9

9 9

9 »99

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; 23.4; 20.8; 18.6; 12.0; MS (LRFAB), NBA + Li) m / z 677 (M + Li) +.

P. Synthesis of dichloro-2,3- (R, R) -cyclohexano-6- (S) - {3- (lithocholylamino) -propyl} 1,4,7,10,13-penta-azacyclopentadecane manganese complex

2,3- (R, R) -cyclohexano-6- (S) - (3- (lithocholylamino) propyl} -1,4,7,10,13pentaazacyclopentadecane prepared as in Example 10 (547 mg, 0.817 mmol) was added to hot anhydrous methanol (50 mL) containing manganese (II) chloride (103 mg, 0.818 mmol) under a dry nitrogen atmosphere. After refluxing for 2 h, the solution was brought to dryness and the residue was dissolved in a mixture of solvents THF (35 mL) and ethyl ether (5 mL) and filtered through a celite pad. Concentration and trituration with 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; Anal. Calcd. for C18H18N3O2N2O: C, 61.79; H, 9.87; N, 10.55; Cl, 8.90. Found: C, 62.67%, 9.84-24, 8.04%, 8.29. - Example 2

Kinetic analysis of the stopped flow method

Stopped flow kinetic analysis has been used to determine whether a compound can catalyze the superoxide dissemination (Riley, DP, Rivers, WJ and Weiss, RH, "Stopped Flow Kinetic Analysis for Monitoring Superoxide Decomposition in Water Systems", Anal. Biochem., 196 344-349 [1991]). To obtain accurate and accurate measurements, all reagents were biologically pure and free of metals. To achieve this, all buffers (Calbiochem) were biologically pure, metal-free and handled with tools that were first washed with 0.1 M HCl followed by purified water, then rinsed in a 10 ⁴ M EDTA bath at pH 8, further rinsed in purified water and dried at 65 ° C for several hours. Dry DMSO solutions of potassium superoxide (Aldrich) were prepared under a dry, inert argon atmosphere in a dry, vacuum atmosphere glove box using dried glassware. DMSO solutions were prepared immediately before each experiment using the stop flow method. A mortar and pestle were used to crush the yellow solid potassium superoxide (about 100 mg). The powder was then triturated with a few drops of DMSO and the slurry was transferred to a flask containing an additional 25 mL of DMSO. The resulting slurry was stirred for 1/2 hour and then filtered. This procedure reproducibly gave about 2 mM superoxide concentration in DMSO. These solutions were transferred to a glove bag under nitrogen in sealed vials prior to filling the syringe under nitrogen. It should be noted that DMSO / superoxide solutions are very sensitive to water, heat, air, and metals. The fresh, clean solution has a slight yellowish tinge.

The buffer solution water was supplied from the proprietary deionized water system to the Bamstead Nanopure Ultrapure Senes 550 aqueous system and then distilled twice, first from basic 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 additionally sodium hydroxide necessary to adjust the pH to 9.0 was added to the corresponding 2 liter flask with distillation head. This distillation oxidizes any traces of organic compounds in the water. The final distillation was carried out under nitrogen in a 2.5 liter flask containing 1500 ml of water from the first distillation and 1.0 x 10 ⁶ MEDTA. This step removes the remaining stems from ultrapure water. To prevent turbidity of the EDTA from volatilizing through the reflux condenser attachment _{, the} 40 cm vertical vertical flask was filled with glass beads and coated with insulation. This system produces oxygen-free water that can have a conductivity of less than 2.0 nanosiemens / cm ² .

The stop flow spectrometer system was designed and manufactured by Kinetic Instruments Inc. (Ann Arbor, Ml) and was connected to a MAC IICX personal computer. Steady-state analysis software was provided by Kinetic Instruments Inc. and was written in QuickBasic with MacAdios drivers. Typical injection volumes (0.10 ml buffer and 0.006 ml DMSO) were calibrated by mixing together a large excess of water with DMSO. The effective ratio was approximately 19/1, so the initial superoxide concentration in the aqueous solution was in the range of 60 to 120 μΜ. Since the published superoxide extinction coefficient in H ₂ O at 245 nm is about 2250 M * ¹ cm ^-1 (1), an initial absorbance value of approximately 0.3 to 0.5 for a cell with a path length of 2 would be expected cm, and this was observed experimentally. Aqueous solutions to be mixed with DMSO superoxide solutions were prepared using 80 mM Hepes buffer, pH 8.1 (free acid + Na form). One of the syringes was filled with 5 ml of DMSO solution while the other was filled with 5 ml of aqueous buffer solution. The complete injection block, mixing device, and the cell of the spectrophotometer were immersed in a thermostatic circulating water bath at 21.0 ± 0.5 ° C.

Before starting to collect data for superoxide degradation, an average baseline was obtained by injecting several doses of buffer and DMSO solutions into the mixing chamber. These doses were averaged and stored as baseline. The first batches collected over a series of runs were with aqueous solutions that did not contain catalyst. This ensures that each series of experiments is capable of producing first order degradation profiles for superoxide without contamination. If the degradations observed after several injections of buffer solution were of second order, manganese (II) complex solutions could be used. Typically, the potential SOD catalyst was monitored over a wide concentration range. Since the initial superoxide concentration after mixing DMSO with aqueous buffer was about 1.2 x 10 ⁴ M, we wanted to use a manganese (II) complex concentration that is at least 20 times lower than the substrate superoxide concentration. Further, we usually followed compounds for SOD activity using a concentration range of 5 χ 10 - ⁷ to 8 x 10 * M. Data obtained from the experiment were imported into a suitable mathematical program (for example, Cricket Graph) to perform standard kinetic-analysis data. The catalytic fast constant for the dismutation of superoxide by the manganese (II) complex of Example 1 was determined from the linear yield of observed rate constants (K, bs) versus the concentration of manganese (II) complexes. The values were obtained from the linear yields of In absorbance at 245 nm against the time of dismutation of manganese (II) complex superoxide, kxat (M ' ¹ sec ^-1 ) of the manganese complex of Example 1 at pH = 8.1 and 21 ° C calculated 0.77 χ 10 ⁺⁷ M 2 sec ^-1 .

The manganese (II) nitrogen-containing macrocyclic ligand complex of Example 1 is a potent catalyst for superoxide dismutation, as can be seen from the k value above.

Claims (17)

PATENT CLAIMS A compound which is a complex represented by formula (I): Wherein -RrRRRRR / rRttRz / RR / RR, R 37 R 4, R 4, R 5, R 5, R e, R e, R e, R 7, Re, R e ,. R 8 R ₉ independently represent alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylaalkyl, cycloalkylcycloalkyl, cycloalkenylalkyl, alkylcycloalkyl, alkenylcycloalkyl, alkylcycloalkenyl, alkenylcycloalkenyl, heterocyclic, aryl and aralkyl and radicals attached to α-carbon α-amino acids; or R 1 or R \ and _R2 or _R'2, R3 or _R'3 and _R4 or _{R'4, R5} or R _'and R _e or R _{6 l} R ₇ or R'? and R ₈ or R ₈ , R ₉ or R ' ₉ and R or R' together with the carbon atoms to which they are attached independently form saturated, partially saturated or unsaturated cycles having 3 to 20 carbon atoms; or R or R 'and R 1 or R 1, R ₂ or R ₂ and R ₃ or R' ₃ , R ₄ or R ' ₄ and R ₅ or R' _s , R ₆ or _in R 'and R ₇ or R ' ₇ , and R _e or R' ₈ and R ₉ or R ' ₉ together with the carbon atoms 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, which does not contain hydrogen bonded to nitrogen, hydrogen bonded to nitrogen in said formula (I), the nitrogen of which is also present 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 "R" groups are attached to the biomolecules 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 z X, Y and Z are attached to the biomolecules via a linking group; and said biomolecules are independently selected from the group consisting of &lt; RTI ID = 0.0 &gt; consisting of &lt; / RTI &gt; steroids, carbohydrates, fatty acids, amino acids, peptides, proteins, antibodies, vitamins, lipids, phospholipids, phosphates, phosphites, nucleic acids, enzyme substrates, enzyme inhibitors and enzyme receptor substrates, and said linker group is derived from a substituent attached to said " R 'or to said X, Y and Z, which reacts with the biomolecule and is selected from the group consisting of -NH ₂ , -NHR ₁₀ , -SH, -OH, -COOH, -COOR ₁₀ , -CONH ₂ , - NCO, -NCS, COOX ', alkenyl, alkynyl, halide, p-toluenesulfonate, methanesulfonate, tresylate, triflate and phenol, wherein R ₁₀ is alkyl, aryl, or alkylaryl and X' is halide; and wherein X, Y and Z are ligands independently selected from the group consisting of halide, oxo, aqua, hydroxo, alcohol, phenol, molecular oxygen, peroxo, hydroperoxo, alkylperoxo, arylperoxo, amine, alkylamine, arylamine, heterocycloalkylamine, heterocycloarylamine, amine oxides, hydrazine, alkylhydrazine, arylhydrazine, nitric oxide, cyano, cyanato, thiocyanato, isocyanato, isothiocyanato, alkyltril, arylnitrile, nitrato, nitrato, azido, alkylsulfonic acid. arylsulphonic acid, alkylsulphoxide, arylsulphoxide, alkylarylsulphoxide, alkylsulphenic acid, arsulphenoic acid, alkylsulfinic acid, arylsulfinic acid, alkylthiol carboxylic acid, arylthiol carboxylic acid, alkylthiolthiocarboxylic acid, arylthiolthiocarboxylic acid, arylthiolthiocarboxylic acid, arylthiolthiocarboxylic acid, arylthiolthiocarboxylic acid, arylthiolthiocarboxylic acid , alkylarylmočovina, thiourea, alkylthiomočovina, arylthiomočovina, alkylarylthiomočovina, sulfate, sulfite, bisulfate, bisulfite, thiosulfate, thiosulfite, hydrosulfite, alkyl phosphine, aryl phosphine, alkyl phosphine oxides, arylfosfinoxid, alkyiarylfosfinoxid, alkylfosfinsulfid, arylfosfinsulfid, alkylarylfosfinsulfid, alkylphosphonic acid, alkylphosphonic acid, Alkyl phosphonium acid arylphosphinic acid, diphenylphosphinic acid, arylphosphinic acid, phosphate, thiophosphate, phosphite, diphosphite, triphosphate, hydrogenphosphate orate, dihydrogen phosphate, alkylguanidino, arylguanidino, alkylarylguanidino, alkylcarbamate, arylcarbamate, alkylarylcarbamate, alkylthiocarbamate, alkylarylthiocarbamate, alkyldithiocarbate, bromo, tetrafluoroborate, hexafluorophosphate, hexafluoroantimonate, phosphonate, iodate, periodate, metaborate, tetraarylborate, tetraalkylborate, tartrate, salicylate, succinate, citrate, ascorbate, saccharinate, amino acid, hydroxamic acid, · thionic acid, or · thionic acid · The corresponding anions, or X, Y and Z are independently bonded to one or more "R" groups and n is 0 or 1. A compound according to claim 1, wherein 1 to 2 "R" groups are attached to the biomolecules via a linking group and none of X, Y and Z is attached to the biomolecule via a linking group. The compound of claim 1, wherein one of X, Y and Z is attached to the biomolecule via a linking group and none of the "R" groups is attached to the biomolecules via a linking group. The compound of claim 1, wherein at most one "R" group attached to the carbon atoms of the macrocycle located between the nitrogen atoms has a biomolecule attached through a linking group. __________________ 5. A compound according to claim 1, _kde_nejméné.jedna-Z- _{R-ri-skupinrkteré} jsou'navázáný.1C biomolecules via a linker group are independently selected from the group consisting of alkyl radicals, cycloalkyl, cycloalkylalkyl, aralkyl, alkaryl, aryl, heterocycles and The hydrocarbon radicals attached to the α-carbon α-amino acids, and the remaining "R" groups are independently selected from hydrogen, a saturated, partially saturated or unsaturated nitrogen-containing cycle or heterocycle. The compound of claim 5, wherein at least two of the "R" groups, further to the "R" groups that are attached to the biomolecules via a linking group, are independently selected from alkyl, cycloalkyl, cycloalkylalkyl, aralkyl, alkaryl, aryl, heterocycles and hydrocarbon residues attached to α-carbon α-amino acids. 7. A compound according to claim 5, wherein at least one of the "R" groups, hereinafter the 'R ^u 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 an unsaturated cycle. A compound according to claim 1, wherein at least one of R ₁ or R '1 and R ₂ or R' ₂ , R ₃ or R ' ₃ and R ₄ or R' 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 ' _g 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 "R" groups, hereinafter "R" groups, which are attached to the biomolecules via linking groups are independently selected from hydrogen, nitrogen-containing heterocycles or alkyl groups. A compound according to claim 8, wherein at least two of 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 ' _s and R _e or R' _e , R7 or R? and R ₈ or R, ^R 8, and R or R '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 The "R" groups, which are attached to the biomolecules via linking groups, are independently selected from hydrogen, nitrogen-containing heterocycles or alkyl groups. - The compound of claim 8, wherein said saturated, partially saturated or unsaturated cyclic residue is cyclohexyl. —_ <—-— The compound of claim 10, wherein said remaining "R" groups, further to the "R groups" that are attached to the biomolecules via linking groups, are independently selected from hydrogen or alkyl groups. The compound of claim 1, wherein said R or R 'a R 1 or R' _b R ₂ or R ' ₂ and R ₃ or R' ₃ R ₄ or R ' ₄ and R ₅ or R', R a or R ' _e and R? or R ' ₇ and R ' ₈ or R ' ₈ and R &apos; ₉ or R &apos; 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 &quot; R &quot; Groups that are attached to the biomolecules via a linking group 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 a halide, an organic acid anion, a nitrate, and a bicarbonate anion. A pharmaceutical composition in unit dosage form useful for dismuturing superoxide, comprising (a) a therapeutically or prophylactically effective amount of the r complex • 9 »0 • 0 0 • 0 « 9 · · · 0 · 9 · 00 ·· 0 · · «• 0 · 9 9 9 0 And (b) a non-toxic, pharmaceutically acceptable carrier, adjuvant or vehicle. A method of preventing or treating a disease or disorder that is mediated, at least in part, by superoxide, comprising administering to a subject in need of such prevention or treatment a therapeutically, prophylactically, pathologically, or resuscitatively effective amount of a complex according to claim 1. The method of claim 15, wherein said disease or disorder is selected from the group consisting of ischemic reperfusion injury, surgically induced ischemia, inflammation of the internal organs, rheumatoid arthritis, atherosclerosis, thrombosis, platelet aggregation, oxidant-induced injury and tissue damage, osteoarthritis , psoriasis, organ transplant rejection, radiation-induced injury, stroke, acute pancreatitis, insulin dependent diabetes mellitus, adult and infantile-respiratory-stress-metastasis, and carcinogenesis. AND. The method of claim 16, wherein said disease or disorder is selected from the group consisting of ischemic reperfusion injury, stroke, atherosclerosis, and inflammatory diseases of internal organs.