JP2006500383A - Peroxynitrite transfer catalyst for treating or preventing diseases caused by peroxynitrite-mediated reactions - Google Patents

Abstract

The present invention relates to paramagnetic 3-, 8 having various substituents at the 13- and 17-positions of the porphyrin skeleton as peroxynitrite transfer catalysts to treat and prevent diseases characterized by radical-mediated cell damage. -Relates to the use of substituted porphyrin derivatives.

Description

  The present invention relates to the use of metal-containing complexes that catalyze peroxynitrite transfer for the manufacture of disease therapeutics.

As early as 1990, peroxynitrite was produced by a diffusion-controlled reaction between nitric oxide (NO, nitric oxide) and the superoxide anion (O ²⁻ ). (Beckman et al., 1990, Proc. Natl. Acad. Sci. USA 87, 1620-1624). Peroxynitrite has been implicated in a number of inflammatory processes that play an important role in diseases such as Alzheimer's dementia, multiple sclerosis, and amyotrophic lateral sclerosis, and is associated with cytopathic and apoptosis induction Cause.

  Peroxynitrite reacts with many proteins through amino acid groups that are oxidized or nitrated. Since peroxynitrite guarantees nitration of tyrosine radicals in motoneuron filaments, nitrotyrosine radicals are often found in the tissues of patients suffering from multiple sclerosis. Neuronal dysfunction (Estevez et al., 1999, Science 286, 2498-2500) is caused by these collapsed contractions of the filament. The cause of vasoconstriction that is impaired after a stroke is the oxidation of cell membrane lipid radicals, induced by peroxynitrite, where the resulting damage to the endothelium and epidermis—and the formation of neutrophils—occurs.

Pharmacological involvement to prevent peroxynitrite-mediated effects can be performed on a portion of the starting material (NO and O ²⁻ ) or a portion of the product.

  The approach for a portion of the product peroxynitrite was first described by Salvemini et al. (Salvemini et al., 1998, Proc. Natl. Acad. Sci. USA 95, 2659-2663). In this approach, peroxynitrite is catalytically transferred to a harmless end product. Large amounts of peroxynitrite can be converted with a slightly lower concentration of catalyst. The advantage of this approach is based on the fact that it cannot produce faulty degradation products, such as reactive oxygen species, and it eliminates inhibition of superoxide dismutase (SOD) by peroxynitrite. Eventually, this treatment using the new compound has a double advantage in treating the disease. Thus, on the one hand, the conversion of peroxynitrite is accelerated and on the other hand, SOD is protected against inactivation by peroxynitrite.

  As possible conversion catalysts, metal-containing complexes are known to date (WO 95/31197, US 6,245,758, WO 98/04132, US 5,872,124, WO 00/75144, WO 01/26655, US 6,372,727). The metalloporphyrins described by Salvemini et al. Are protective in inflammation models (Salvemini et al., 1998, Proc. Natl. Acad. Sci. USA 95, 2659-2663 and Brit. J. Pharmacol., 1999, 127, 685-692. ). Cuzzocrea et al. Described that the same class of compounds was effective in models of iliac occlusion (Cuzzocrea et al., 2000, FASEB J. 14 (9), 1061-1107 and Cuzzocrea et al., 2001, Pharmacology Rev. 53, 135- 159). Cross et al. Demonstrated the effectiveness of these substances in a mouse MS model ("Experimental Autoimmune Encephalomyelitis" = EAE) (Cross et al., 2000, J. Neuroimmunology 107, 21-28). Mackensen et al. First demonstrated the effectiveness of manganese-containing porphyrins in a localized ischemic model, localized MCAO (central cerebral artery occlusion) (Mackensen et al., 2001, J. Neurosci. 21, 4582-4582).

To date, little is known about side effects such as toxicity and in vivo efficacy, and the blood-brain spinal permeability of these known translocation catalysts.
In order to treat and prevent causative diseases in reactions mediated by peroxynitrite, an urgent issue is well-tolerated that is effective in vivo to increase the transfer of peroxynitrite in harmless products. To produce a chemically stable substance (catalyst). These substances that are effective in vivo can be used to develop drugs to treat diseases.

  The present invention solves this problem by producing a porphyrin complex used as a peroxynitrite transfer catalyst. These porphyrins are distinguished by having excellent in vivo efficacy and chemical stability. They have already been used as substances for diagnosing tumors, and their use in necrosis and infarct imaging has already been disclosed in WO 00/05235.

  In the present invention, NMR and UV / VIS spectroscopy could show that according to the present invention the porphyrin of WO 00/05235 catalyzes the transfer of peroxynitrite in the harmless end products, namely nitrate and nitrite. . A model of cell damage could show that the porphyrin according to the present invention is protective and protects the cells from peroxynitrite damage induced by the peroxynitrite donor SIN-I. These porphyrins are very well characterized, and they are known to have no side effects, have good water solubility and good in vivo efficacy.

The use of porphyrin complexes, which have peroxynitrite transfer properties on the one hand and diagnostic properties on the other hand, allow for special treatment of diseases caused by peroxynitrite and diagnosis using imaging methods such as MRT. Become.
The present invention provides this special therapeutic porphyrin complex and consists of a ligand of general formula I below and at least one ion of an element of atomic number 20-32, 37-39, 42-51 or 57-83 About:

Where
M means a paramagnetic ion;
R ¹ represents a hydrogen atom, a linear C ₁ -C ₆ alkyl group, a C ₇ -C ₁₂ aralkyl group or a group OR ′, wherein R ′ is a hydrogen atom or a C ₁ -C ₃ alkyl group;
R ² means R ³ , the group -CO-Z or the group-(NH) _o- (A) _q -NH-D, where Z is the group -OL, where L is an inorganic or organic cation Or C ₁ -C ₄ alkyl group, A means phenyleneoxy group or C ₁ -C ₁₂ alkylene group or C ₇ -C ₁₂ aralkylene group (interrupted by one or more oxygen atoms) , O and q, independently of one another, denote the number 0 or 1, and D denotes a hydrogen atom or the group --CO-A- (COOL) _o- (H) _m , where m is 0 or 1 Equal, but the sum of m and o is equal to 1;

R ³ represents a group-(C = Q) (NR ⁴ ) _o- (A) _q- (NR ⁵ ) -K, where Q represents an oxygen atom or two hydrogen atoms, and R ⁴ represents Means the group-(A) _q -H, and K means a complexing agent of the general formula (IIa), (IIb), (IIc), (IId) or (IIe), wherein R ⁵ is When K is a complexing agent of formula (IIa), it has the same meaning as R ⁴ and R ⁵ is a complexing of K of formula (IIb), (IIc), (IId) or (IIe) In the case of an agent, it has the same meaning as D, but no direct oxygen-nitrogen bond is possible, and K is represented by the general formulas (IIa), (IIb), (IIc), (IId), (IIe) ) Or (IIf) complexing agent,

Where
q has the meaning indicated above;
A ¹ has the meaning indicated for A;
R ⁶ represents a hydrogen atom, a linear or branched C ₁ -C ₇ alkyl group, a phenyl group or a benzyl group;

A ² is phenylene, -CH ₂ -NHCO-CH ₂ -CH (CH ₂ COOH) -C ₆ H ₄ -β-, -C ₆ H ₄ -O- (CH ₂ ) _0-5 -β, or -C _{6 H 4 - (OCH 2 CH} 2) 0-1 -N (CH 2 COOH) means -CH _2-beta group or C ₁ -C ₁₂ alkylene or C ₇ -C ₁₂ alkylene group, said here The alkylene group may be substituted by one or more oxygen atoms, 1 to 3 -NHCO groups, or 1 to 3 -CONH groups and / or 1 to 3-(CH _{2 0-5} optionally substituted by a COOH group, where β means the binding site for X;
X represents a —CO— or NHCS group;

L ¹ , L ² , L ³ and L ⁴ independently of each other represent a hydrogen atom or at least one metal ion equivalent of an element of the aforementioned atomic number, provided that at least two of these substituents are metals Means an ionic equivalent, and free carboxylic acid groups, where other anions are present, which are not required for complexation, are physiologically compatible to compensate for the charge that is optionally present in the metalloporphyrin. Can be present as a salt with an inorganic and / or organic cation, as an ester, or as an amide,
Moreover, the porphyrin complex can be used in the manufacture of a medicament that increases the transfer rate of peroxynitrite in harmless products, thus treating and preventing radical mediated cell damage.

The compounds according to the invention could be shown by NMR and UV / VIS spectroscopy to catalyze the rearrangement of peroxynitrite in harmless end products, namely nitrates and nitrites. Peroxynitrite is a strong oxidant produced by the reaction of nitric oxide (NO) with a superoxide anion (O ²⁻ ). NO could be shown to occur in many cells, eg macrophages, neutrophil cells, hepatocytes and endothelial cells. Direct reaction of NO with superoxide anion (O ²⁻ ) forms a peroxynitrite ion, which dissolves rapidly in oxidative intermediates under physiological conditions. These intermediate oxidation stages cause damage to biological targets.

  The consequences of this damage can be related to pathological consequences including the oxidation and nitration of proteins, lipids and DNA. Peroxynitrite passes through the cell membrane at a significantly higher rate than other oxidants and can penetrate rapidly into the cell even in the presence of biological membranes. Peroxynitrite is known for nitration of tyrosine radicals in proteins and oxidizes sulfhydryl radicals, methionine and macromolecules such as metalloenzymes, DNA and lipids.

  Since peroxynitrite is highly reactive, it has been contacted with a number of diseases. The present invention relates to the use of the compounds according to the invention for the manufacture of a medicament for treating and preventing radical-mediated cell damage. These include neurodegenerative diseases, inflammatory diseases, autoimmune diseases, and cardiovascular diseases.

For example, the following can be stated:
Other neurodegenerative diseases associated with cerebral ischemia, ischemic reperfusion disease, hypoxia and inflammation, such as multiple sclerosis, ALS (amyotrophic lateral sclerosis) and comparable sclerosis disease, Parkinson Disease, Huntington's disease, Korsakov's disease, epilepsy, vomiting, hypnosis, schizophrenia, depression, stress, pain, migraine, hypoglycemia, dementia such as Alzheimer's disease, HIV dementia and presenile dementia.

  They also treat cardiovascular diseases such as atherosclerosis, and autoimmune and / or inflammatory diseases such as hypotension, ARDS (adult respiratory distress syndrome), sepsis or septic Shock, rheumatoid arthritis, osteoarthritis, insulin-dependent diabetes mellitus (IDDM), pelvic / intestinal inflammatory disease (enteric disease), meningitis, glomerulonephritis, acute and chronic liver disease, rejection disease ( Suitable for the treatment of allogeneic heart, kidney or liver transplantation) or inflammatory skin diseases such as psoriasis, and others.

The porphyrin complex according to the present invention contains iron (III), manganese (III), copper (II), cobalt (III), chromium (III), nickel (II) or vanadyl (II) as paramagnetic ions in the porphyrin skeleton. The first three ions that contain ions and are described here are preferred.
If one of the ions bound in the porphyrin is present in an oxidation step higher than +2, one or more excess charges may be caused by, for example, an anion of an organic or inorganic acid, preferably acetate, chloride, Compensated by ions of the product, sulfate, nitrate, tartrate, succinate and maleate or by the negative charge present in R ² and / or R ³ .

  Carboxyl groups that are unnecessary for complexing metal ions can be present as esters, amides, or salts of inorganic or organic bases as required. Suitable ester groups are groups having 1 to 6 C atoms, preferably ethyl esters; suitable inorganic cations are, for example, lithium ions and potassium ions, in particular sodium ions. Suitable cations of the organic base are primary, secondary or tertiary amines such as ethanolamine, diethanolamine, morpholine, glucamine, N, N-dimethylglucamine, in particular meglumine cation.

As the complexing agent group K, preferably diethyltriaminepentaacetic acid and 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid derivatives can be listed, which are linked via a linker. To each porphyrin.
The preparation of complex compounds of the general formula I is carried out according to methods known in the literature (for IIa and IIb see eg DE 4232925; for IIIc see eg DE 19507822, DE 19580858 and DE 19507819; and IId, (For example, see US 5,053,503, WO 96/02669, WO 96/01655, EP 0430863, EP 255471, US 5,277,895, EP 0232751, and US 4,885,363 for IIe and IIf).

The preparation of the compounds according to the invention has already been described in WO 00/17205.
Compounds in which R ² and R ³ represent a CONHNHK group are preferred. The synthesis of 3,3 '-(7,12-diethyl-3,8,13,17-tetramethylporphyrin-2,18-diyl) di (propanohydrazide) required as an educt in this regard is described in Z. Physiol Chem. 241, 209 (1936).

The introduction of the required metal (for example Mn) into the porphyrin is carried out according to methods known in the literature (for example The Porphyrins, Editor D. Dolphin, Academic Press, New York 1980, Vol. V, 459; DE 4232925), where essentially the following methods can be listed:
a) Substitution of pyrrole NH (heat the metal-free ligand in a polar solvent by adding the corresponding metal salt, e.g. acetate, and optionally an acid buffer, e.g. sodium acetate), or
b) “Complexation”, where the metal already complexed by the ligand is replaced with the required metal.

As solvents, mainly polar solvents, for example, methanol, glacial acetic acid, dimethylformamide, chloroform and water are suitable.
The introduction of the paramagnetic metal M into the porphyrin system can be carried out before or after the binding of the complexing agent group K. In the end, a particularly flexible procedure for synthesizing the compounds according to the invention is possible.
The chelation of the group K is carried out according to methods known in the literature (see for example DE 34 01 052), where the metal oxide or metal salt of the respective required metal (for example nitrate, carbonate, chloride). Product or sulfate) is suspended or dissolved in a polar solvent such as water or aqueous alcohol and reacted with the corresponding amount of complexing ligand. If necessary, the acidic hydrogen atoms or acid groups present can be replaced by inorganic bases and / or organic bases or amino acid cations.

  In this case, the neutralization is carried out with an inorganic base such as an alkali metal or alkaline earth metal hydroxide, carbonate or bicarbonate and / or an organic base such as, among others, primary, secondary and tertiary. Of secondary amines such as ethanolamine, morpholine, glucamine, N-methylglucamine and N, N-dimethylglucamine, and basic amino acids such as lysine, arginine and ornithine, or amides of naturally neutral or acidic amino acids Implemented with help.

  In order to produce a neutral complex compound, sufficient necessary base can be added, for example to an acidic complex salt in an aqueous solution or suspension, so as to reach a neutral point. The resulting solution can then be evaporated to dryness in vacuo. Formed by adding a water miscible solvent, for example, a lower alcohol (e.g., methanol, ethanol, isopropanol), a lower ketone (e.g., acetone), a polar ether (e.g., tetrahydrofuran, dioxane, 1,2-dimethoxyethane) It is often convenient to precipitate neutral salts and thus easily isolate and purify the crystals. It has proved particularly advantageous to add the necessary base as early as during the complexation of the reaction mixture, thus saving process steps.

If the acidic complex compound contains several free acid groups, it is often appropriate to produce a neutral mixed salt containing both inorganic and organic cations as counterions.
This can be performed, for example, as follows. That is, a complexing ligand is reacted in an aqueous suspension or solution with an oxide or salt of an element that generates a central ion and an organic base required for neutralization in half the amount, and the formed complex salt is isolated and necessary. Purify accordingly and then mix with the amount of inorganic base required for complete neutralization. Also, the order of base addition can be reversed.
The possibility of forming a neutral complex compound consists in completely or partially converting the remaining acid groups in the complex into esters. This can be done by subsequent reaction in the finished complex (eg by thoroughly reacting the free carboxy group with dimethyl sulfate).

  Moreover, manufacture of the chemical | medical agent by this invention is implemented as follows according to the method known in this field | area. That is, the complex compound according to the present invention is suspended or dissolved in an aqueous medium by adding additives commonly used in crude drugs as necessary, and then the suspension or solution is sterilized as necessary. . Suitable additives include, for example, physiologically harmless buffers (e.g. tromethamine), small amounts of complexing agents (e.g. diethylenetriaminepentaacetic acid), or, if necessary, electrolytes such as sodium chloride or as required Depending on the antioxidant, for example ascorbic acid.

In principle, it is also possible to produce the medicament according to the invention without isolating the complex salt. In each case, care must be taken to carry out the chelation so that the salts and salt solutions according to the invention are virtually free of toxic non-complexed metal ions.
This can be ensured, for example, by controlled titration during the manufacturing process with the aid of a color indicator such as xylenol orange. Therefore, the present invention also relates to a method for producing complex compounds and salts thereof. As a final preventive measure, purification of the isolated complex remains.

  In order to use the compounds according to the invention as medicaments, the compounds according to the invention may be combined with active ingredients for enteral or parenteral administration, in addition to suitable pharmaceutically organic or inorganic inert carrier substances, for example It is in the form of a pharmaceutical formulation containing water, gelatin, gum arabic, lactose, starch, magnesium stearate, talc, vegetable oil, polyalkylene glycol, and others. The pharmaceutical preparation can be present in solid form, for example in the form of tablets, coated tablets, suppositories or capsules, or in liquid form, for example solutions, suspensions or emulsions. In addition, the pharmaceutical formulation optionally contains an adjuvant, such as a preservative, stabilizer, wetting or emulsifying agent; osmotic pressure modifier, or buffer.

These pharmaceutical preparations are also the subject of the present invention.
For parenteral use, injectable solutions or suspensions, especially aqueous solutions of the active compounds in polyhydroxyethoxylated castor oil are suitable.
Where a suspension or solution of a compound according to the present invention in water or saline solution is desired for enteral administration or other purposes, the compound according to the present invention may be combined with one or more adjuvants commonly used in herbal medicines ( For example, methylcellulose, lactose, mannitol) and / or one or more surfactants (eg, lecithin, Tween ™, Myrj ™) and / or flavoring agents for taste correction (eg, ethers) Oil).

As carrier systems, surface-active adjuvants such as bile salts or animal or vegetable phospholipids, but also mixtures thereof, as well as liposomes or components thereof, can also be used.
For oral administration, tablets, coated tablets or capsules containing talc and / or hydrocarbon excipients or binders, such as lactose, corn or potato starch, are particularly suitable. It can also be applied in liquid form, for example juice, with sweeteners added as needed.

Intestinal, parenteral and oral administration are also the subject of the present invention.
The dosage of the active ingredient can be varied depending on the mode of administration, the age and weight of the patient, the type and severity of the disease to be treated. The daily dose is 0.5 to 1000 mg, preferably 50 to 200 mg, where the dose can be administered as a single dose to be administered at one time, or two or more daily doses Can be divided for.
The invention also provides the use of a porphyrin complex of formula (I) according to the invention for treating and preventing diseases caused by peroxynitrite-mediated reactions and attenuated and / or treated by increasing the conversion rate of peroxynitrite About.

The invention relates in particular to the use of the porphyrin complex of general formula (I) according to the invention for the treatment and prevention of diseases including neurodegenerative diseases, inflammatory diseases, autoimmune diseases and cardiovascular diseases. For example, the following diseases can be listed:
-Other neurodegenerative diseases associated with cerebral ischemia, ischemic reperfusion disease, hypoxia and inflammation, such as multiple sclerosis, amyotrophic lateral sclerosis and comparable sclerosis disease, Parkinson's disease, Huntington's disease, Korsakov syndrome, epilepsy, vomiting, hypnosis, schizophrenia, depression, migraine, hypoglycemia, dementia such as Alzheimer's disease, HIV dementia and presenile dementia.

-They also treat cardiovascular diseases and autoimmune and / or inflammatory diseases such as hypotension, ARDS (adult respiratory distress syndrome), sepsis or septic shock, rheumatoid arthritis, bone Arthritis, insulin-dependent diabetes mellitus (IDDM), pelvic / intestinal inflammatory disease (intestinal disease), meningitis, glomerulonephritis, acute and chronic liver disease, rejection-related disease (eg, allogeneic heart Suitable for the treatment of inflammatory skin diseases such as psoriasis, and other diseases. Based on the structure of action, the compounds according to the invention are very well suited for the rearrangement of peroxynitrite in harmless products.
The subject of the invention is also that M means Fe ³⁺ , Mn ³⁺ , Cu ²⁺ , Co ³⁺ , VO ²⁺ , Cr ³⁺ or Ni ²⁺ ions, and is particularly effective. The use of a compound of general formula (I) is characterized.

Further, the subject of the present invention is that in each case R ² and R ³ are -CONHNHK, -CONH (CH ₂ ) ₂ NHK, -CONH (CH ₂ ) ₃ NHK, -CONH (CH ₂ ) ₄ NHK, or -CONH Use of a porphyrin complex compound of the general formula I, characterized in that it means a (CH ₂ ) ₂ O (CH ₂ ) ₂ NHK group.
The subject of the invention is also the use of a porphyrin complex compound of the general formula (I), characterized in that R ² and R ³ in each case mean —CONHNHK.
In these compounds, K is represented by the general formula (IIa)

の錯化剤である場合、非常に特に有効である。

It is very particularly effective when it is a complexing agent.

Additional subjects of the invention are in particular porphyrin complex compounds of the formula (I), i.e.
{mu-[{16,16 '-[Chloromanganese (III) -7,12-diethyl-3,8,13,17-tetramethylporphyrin-2,18-diyl] -bis [3,6,9- Tris (carboxymethyl) -11,14-dioxo-3,6,9,12,13-pentaazahexadecanoato]} (8-)]} digadolinato (2-), disodium,
{mu [{16,16 '-[Chloroiron (III) -7,12-diethyl-3,8,13,17-tetramethylporphyrin-2,18-diyl] -bis [3,6,9-tris (Carboxymethyl) -11,14-dioxo-3,6,9,12,13-pentaazahexadecanoato]} (8-)]} digadolinato (2-), disodium,
{mu [{16,16 '-[Copper (II) -7,12-diethyl-3,8,13,17-tetramethylporphyrin-2,18-diyl] -bis [3,6,9-tris ( Carboxymethyl) -11,14-dioxo-3,6,9,12,13-pentaazahexadecanoato]} (8-)]} digadolinato (2-), disodium,
It is.

  Since the compounds according to the invention have excellent water solubility, it is possible to prepare highly concentrated solutions, keep the volumetric load of the circulatory system within reasonable limits, and compensate for dilution with body fluids. Furthermore, the compounds according to the invention not only have high in vitro stability, but also surprisingly high in vivo stability, so that they are inherently toxic within the time required for complete elimination of the contrast agent again. The release or exchange of ions that are present and not covalently bound to the complex can be ignored.

  Surprisingly enough, the complexes according to the invention show a significantly higher relaxivity compared to the conventionally known structurally similar compounds. Since relaxivity can be regarded as a measure of the contrast effect of the compound, comparable positive signal effects are possible, even at low doses, using the compounds according to the invention in the NMR diagnostic domain. This significantly increases the safety factor, where the product of relaxation and fitness can be considered an index value.

  Furthermore, the present invention relates to the use of a porphyrin complex of general formula I according to claim 1 for the treatment and prevention of diseases comprising the following groups of diseases: ischemic reperfusion diseases such as stroke, head trauma And myocardial infarction, sepsis, chronic or acute inflammation (eg arthritis or inflammatory bowel disease), adult respiratory stress syndrome, cancer, broncho-pulmonary dysplasia, cardiovascular disease, diabetes, multiple sclerosis, Parkinson's disease, Familial amyotrophic lateral sclerosis and colitis and special neurological diseases.

1. Study of peroxynitrite degradation using NMR spectroscopy For this study, three samples were prepared, where one of the samples contained the original aqueous peroxynitrite solution without additives and the other The sample contains the same amount of this solution, but contains a defined addition amount of reference material or material to be studied. From each sample, ¹⁴ N spectra are recorded using the same acquisition and processing parameters. The difference in the integral of the nitrate signal between the treated peroxynitrite solution and the untreated peroxynitrite solution indicates an increase in nitrate due to the transfer of peroxynitrite. This process can be quantified by comparing the data between the substance to be studied and the reference compound (Figure 1).

2. Measurement of conversion rate of peroxynitrite to nitrate by UV spectroscopy The following were used:
UV spectrometer Flowing device with plug with flanged cuvette Laboratory device for measuring volume Reagents (buffer, peroxynitrite solution, catalyst)
The content of peroxynitrite was determined from the concentrated peroxynitrite solution. As the baseline, a molar extinction coefficient of ε = 1670 is employed. The solution was diluted with water so that the absorption at an observation wavelength of 301 nm reached about 1.6. The pH of the stock solution thus prepared does not drop below 11.

Corresponding to the set concentration of peroxynitrite, in the phosphate buffer so that the required amount of catalyst solution and peroxynitrite can join and generate a reaction, taking into account the predetermined ratio of the plugged flow device Prepare the catalyst solution to be studied. The catalyst solution buffer must still have sufficient capacity to set and maintain the strong alkaline peroxynitrite solution at the required pH. Add catalyst in 100x deficiency.
Fill the metering nebulizer of the plugged flow device with the two solutions and place the solution from the metering nebulizer into the cuvette located in the UV spectrometer. The absorption value is measured simultaneously at 301 nm. Due to the rearrangement of peroxynitrite in nitrate, the absorption decreases and takes a stable value after a certain time. At this point, the dislocation stops and data recording stops.

The measured data can be analyzed and the peroxynitrite concentration can be calculated from the kinetic characteristics of the resulting curve. These data are used to characterize the catalyst to be studied. To take into account the self-decomposition of peroxynitrite, a catalyst-free buffer is added to first determine the decomposition behavior of the peroxynitrite solution in the peroxynitrite preparation used and from measurements using the catalyst. For the properties to be obtained, set those properties.
Relative rate constant = Spontaneous degradation of peroxynitrite used at the stated pH for catalyzed degradation
(Fig. 2).

3. SIN-1 injury assay using primary neuronal cultures from neonatal rat cerebellum: Primary culture of cells from neonatal rat cerebellum for in vitro testing of neuroprotective substances for peroxynitrite-induced damage Apply things. Measurement of the death or survival of neuronal cells in this culture is performed indirectly by measuring the response of the diluted fluorescent form of Alamar blue dye. For damage, use the peroxynitrite donor SIN-1 (3-morpholino-sidnonimine).

  To obtain cells, Wistar rats (P8) were killed by decapitation, the cerebellum was acquired, the meninges were removed from the cerebellum (HBSS (GIBCO, 14025-050) 4 ° C), crushed, and 15 ml Falcon Transfer to a (Falcon) tube and aspirate the supernatant, then trypsinize the cerebellum by adding 500 μl trypsin-EDTA solution (GIBCO # 2530-054) / cerebellum. After incubation (20 min, 37 ° C.), trypsinized cerebellum is washed 3 times with 10 ml HBSS. Then titration is performed by adding 500 μl of 0.05% DNAsel (BOEHRINGER MANNHEIM, # 14953000) / cerebellum.

Isolate the cells with a 5 ml pipette, then use a flame polished Pasteur pipette, and finally (if necessary) using an elongated, flame polished Pasteur pipette and add 10 ml complete medium (100 ml Neural base (GIBCO # 21103-049), 1 ml B27 supplement (GIBCO # 17504-044), 0.4 ml Pen / Strep (10,000 IU / ml / 10000 UG / ml) (GIBCO # 15140-106), 0.8 Mix with ml KCl-stock solution (MERCK, 1.04936.0500) and 1 ml L-glutamine (100 x -200 mmol) (GIBCO # 15140-106) .The isolated cells are then centrifuged (at 600 rpm) 10 minutes), washed once with complete medium, resuspended in 20 ml complete medium, counted and diluted to 2 × 10 ⁶ / ml.

  For each well of a 96-well microtiter plate, 100 μl of complete medium is introduced and mixed with 100 μl of cell suspension (= div 1 = day 1 in vitro). Pre-coat microtiter plates as follows: 50 μl / well of poly-L-lysine (molecular weight 70 to 105 kD) (SIGMA # P-6282) is applied, then the plate is incubated in an incubator for about 90 minutes To do. Before flattening the cells, aspirate the solution again and wash twice with HBSS or sterile double distilled water. 24 hours after flattening, SIN-1 is added to damage the cells.

One hour before the addition of SIN-1, apply the test substance (individual concentrations of 10 or 30 μM, or as a concentration series, CALBIOCHEM 567028). Cell function is measured for div 2 (day 2 in vitro) using Alamar Blue (10 μl / well) (BIOSOURCE INT., DAL 1100). After 3 hours of incubation, the measurement is performed with a fluorescence reader (Victor, Wallac Company, absorbance 544 nm / emission 590 nm). Calculate IC50 values using Excel Plug-in XL fit.
The results from Examples 2 and 3 are shown in the table below.

FIG. 1 shows the ¹⁴ NMR spectrum. FIG. 2 shows the time-dependent degradation of peroxynitrite in the presence and absence of porphyrin catalyst administered at 100 × deficiency.

Claims (9)

Porphyrin comprising a ligand of the following general formula I and at least one ion of an element having an atomic number of 20-32, 37-39, 42-51 or 57-83 for the manufacture of a medicament for treating or preventing radical-mediated cell damage Use of complex:

Where
M stands for paramagnetic ion,
R ¹ represents a hydrogen atom, a linear C ₁ -C ₆ alkyl group, a C ₇ -C ₁₂ aralkyl group or a group OR ′, wherein R ′ is a hydrogen atom or a C ₁ -C ₃ alkyl group,
R ² means R ³ , the group -CO-Z or the group-(NH) _o- (A) _q -NH-D, where Z is the group -OL, where L is an inorganic or organic cation or refers to C ₁ -C ₄ alkyl group, a is meant phenylene group or C ₁ -C ₁₂ alkylene group or one or more C ₇ -C ₁₂ aralkylene group which is interrupted by oxygen, o and q independently of each other represents the number 0 or 1, and D represents a hydrogen atom or the group --CO-A- (COOL) _o- (H) _m , where m is equal to 0 or 1, provided that The sum of m and o is equal to 1,
R ³ represents a group-(C = Q) (NR ⁴ ) _o- (A) _q- (NR ⁵ ) -K, where Q represents an oxygen atom or two hydrogen atoms, and R ⁴ represents a group -(A) _q -H, and K means a complexing agent of the general formula (IIa), (IIb), (IIc), (IId) or (IIe), wherein R ⁵ is K Is a complexing agent of the formula (IIa) and has the same meaning as R ⁴ and R ⁵ is a complexing agent of the formula (IIb), (IIc), (IId) or (IIe) where K is And has the same meaning as D, but no direct oxygen-nitrogen bond is possible, and K is of the general formula (IIa), (IIb), (IIc), (IId), (IIe) Or (IIf) complexing agent,

Where
q has the meaning given above,
A ¹ has the meaning indicated for A,
R ⁶ represents a hydrogen atom, a linear or branched C ₁ -C ₇ alkyl group, a phenyl group or a benzyl group,
A ² is phenylene, -CH ₂ -NHCO-CH ₂ -CH (CH ₂ COOH) -C ₆ H ₄ -β-, -C ₆ H ₄ -O- (CH ₂ ) _0-5 -β, or -C _{6 H 4 - (OCH 2 CH} 2) 0-1 -N (CH 2 COOH) means -CH _2-beta group or C ₁ -C ₁₂ alkylene group or C ₇ -C ₁₂ alkylene group, said here C ₇ -C ₁₂ alkylene group may be interrupted by one or more oxygen atoms, 1 to 3 —NHCO groups, or 1 to 3 —CONH groups, and / or 1 to 3 -(CH ₂ ) _0-5 optionally substituted by a COOH group, where β means the binding site for X;
X means -CO- or NHCS- group,
L ¹ , L ² , L ³ and L ⁴ independently of each other represent a hydrogen atom or at least one metal ion equivalent of an element of the aforementioned atomic number, provided that at least two of these substituents are metals Means an ionic equivalent, and free carboxylic acid groups, where other anions are present, which are not required for complexation, are physiologically compatible to compensate for the charge that is optionally present in the metalloporphyrin. It can also be present as a salt with an inorganic and / or organic cation, as an ester, or as an amide.   Use of a porphyrin complex according to claim 1 for the manufacture of a medicament for treating and preventing a disease caused by a peroxynitrite-mediated reaction and attenuated and / or treated by increasing the conversion rate of peroxynitrite.   Use of a porphyrin complex according to claim 1 or 2 for treating and preventing diseases including the following groups of diseases: ischemic reperfusion diseases such as stroke, head trauma and myocardial infarction, sepsis, chronic or acute Inflammation (eg, arthritis or inflammatory bowel disease), adult respiratory stress syndrome, cancer, broncho-pulmonary dysplasia, cardiovascular disease, diabetes, multiple sclerosis, Parkinson's disease, familial amyotrophic lateral sclerosis And colitis and special neurological diseases. 2. Porphyrin of general formula I according to claim 1, characterized in that M means Fe3 ⁺ , Mn3 ⁺ , Cu2 ⁺ , Co3 ⁺ , VO2 ⁺ , Cr3 ⁺ or Ni2 ⁺ ions Use of complexes. In each case R ² and R ³ are -CONHNHK, -CONH (CH ₂ ) ₂ NHK, -CONH (CH ₂ ) ₃ NHK, -CONH (CH ₂ ) ₄ NHK, or -CONH (CH ₂ ) ₂ O (CH ₂ ) Use of a porphyrin complex of the general formula I according to claim 1 or 4, meaning ₂ NHK group. Use of a porphyrin complex of general formula I according to claim 1, 4 or 5, wherein R ² and R ³ in each case mean -CONHNHK. K is the general formula (IIa)

7. The compound according to claim 6, which is a complexing agent. Porphyrin complex compound of formula I according to claim 1, i.e.
{mu-[{16,16 '-[Chloromanganese (III) -7,12-diethyl-3,8,13,17-tetramethylporphyrin-2,18-diyl] -bis [3,6,9- Tris (carboxymethyl) -11,14-dioxo-3,6,9,12,13-pentaazahexadecanoato]} (8-)]} digadolinato (2-), disodium,
{mu [{16,16 '-[Chloroiron (III) -7,12-diethyl-3,8,13,17-tetramethylporphyrin-2,18-diyl] -bis [3,6,9-tris (Carboxymethyl) -11,14-dioxo-3,6,9,12,13-pentaazahexadecanoato]} (8-)]} digadolinato (2-), disodium,
{mu [{16,16 '-[Copper (II) -7,12-diethyl-3,8,13,17-tetramethylporphyrin-2,18-diyl] -bis [3,6,9-tris ( Carboxymethyl) -11,14-dioxo-3,6,9,12,13-pentaazahexadecanoato]} (8-)]} digadolinato (2-), disodium,
Use of. Use of general formula I according to claim 1 for diagnosing diseases comprising the following groups:
a. ischemic reperfusion disease,
b. Acute and chronic inflammatory diseases,
c. autoimmune diseases,
d. Neurodegeneration and nerve regeneration diseases.

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