WO1993000326A1 - Omega-3 polyunsaturated fatty-acid derivatives, their preparation and their use - Google Patents
Omega-3 polyunsaturated fatty-acid derivatives, their preparation and their use Download PDFInfo
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- WO1993000326A1 WO1993000326A1 PCT/EP1992/001337 EP9201337W WO9300326A1 WO 1993000326 A1 WO1993000326 A1 WO 1993000326A1 EP 9201337 W EP9201337 W EP 9201337W WO 9300326 A1 WO9300326 A1 WO 9300326A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C219/00—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton
- C07C219/02—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
- C07C219/04—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
- C07C219/08—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having at least one of the hydroxy groups esterified by a carboxylic acid having the esterifying carboxyl group bound to an acyclic carbon atom of an acyclic unsaturated carbon skeleton
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C233/00—Carboxylic acid amides
- C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
- C07C233/34—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups
- C07C233/35—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
- C07C233/38—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to a carbon atom of an acyclic unsaturated carbon skeleton
Definitions
- Omega-3 polyunsaturated fatty acid derivatives their production and use
- the present invention relates to new omega-3 polyunsaturated fatty acid derivatives, their preparation and their use for the treatment of inflammatory processes and septic shock.
- EPA Eicosapentaenoic acid
- DHA docosahexaenoic acid
- one of the mechanisms of action of these fatty acids is due to the influence on the arachidonic acid cascade.
- an EPA diet leads to an increased formation of the intermediate product leukotriene B5 with a simultaneous reduction in leukotriene B4. the latter has a significantly higher inflammatory potential than leukotriene B 5 [J. Clin. Invest. j " 4 1922 (1984); J. Biol. Chem. 259, 7615 (1984)]; likewise the endogenous formation of the platelet activating factor is reduced [Biochem. Biophy
- EPA and DHA A number of other functions are influenced by EPA and DHA.
- the inhibition of the release of mediators from monocytes (tumor necrosis factor, interleukin 1) was described in animals and human subjects after a sufficiently long diet [N. Engl. J. Med. 320, 265 (1989)]; platelet aggregation is also inhibited by EPA [Nephron 43, 196 (1986)].
- EPA has a dampening effect on the proliferation of lymphocytes; moreover, it inhibits the release of lytic enzymes, oxygen radicals and the adherence of granulocytes to the endothelium [N. Engl. J. Med. 312, 1217 (1985); Biochem. Biophys. Res. Comm. 137, 10094 (1986)].
- inhibitory effects can be of therapeutic use in a wide variety of clinical pictures; in particular in acute and chronic inflammatory processes [J. Immunol. 134, 1914 (1985)], rheumatoid diseases [J. Rheumatol. 1_5_, 1471 (1988)], thrombosis and infarction, psoriasis [Lancet Vol.l, 378 (1988)] and septic shock.
- a protective effect in graft versus host disease is also expected.
- the fatty acid ester must be administered intravenously in order to achieve a quick protective effect. It has recently been shown that an effective reduction of LTB4 can be achieved as early as 6 hours after iv administration when eicosapentaenoic acid triclyceride is used [Abstract 7th Intern. Conf. on
- the invention relates to compounds of the formula I.
- Ri is the acyl residue of 5,8, 11, 14, 17-eicosapentaenoic acid
- X is an oxygen atom or an NH group
- Y is a C2-C3 alkylene group
- R2 and R3 are hydrogen atoms or C ⁇ _2 alkyl groups
- X is preferably an oxygen atom
- Y is preferably a C2-alkylene group
- R2 and R3 are preferably methyl or ethyl radicals, but especially methyl radicals.
- the compounds are preferably in the form of salts with physiologically tolerable acids.
- physiologically acceptable acids hydrochloric acid, citric acid, tartaric acid, lactic acid, phosphoric acid, methanesulfonic acid, acetic acid, formic acid, maleic acid, fumaric acid, malic acid, succinic acid, malonic acid, sulfuric acid, L-glutamic acid, L-aspartic acid, pyruvic acid - acid, mucic acid, benzoic acid, glucuronic acid, oxalic acid,
- Ascorbic acid acetylglycine, orotic acid.
- Preferred salts are the hydrochlorides and the trialkylaunonium chlorides.
- the compounds according to the invention can be prepared by general methods for the synthesis of carboxylic acid esters or carboxylic acid amides and, if appropriate, subsequent ammonium salt formation.
- the synthesis of the carboxylic acid esters or carboxamides is preferably the reaction of the carboxylic acid chlorides with the corresponding alcohols or amines in the presence of tertiary amine bases, for example 4-dimethylaminopyridine, or the reaction of the carboxylic acids with the corresponding alcohols or amines in the presence of dehydrating agents , for example dicyclohexylcarbodimide with 4-dimethylaminopyridine as an activator.
- the ester or amide bonds are formed at temperatures from -10 to 100 ° C., preferably at temperatures from 0 to 40 ° C. in inert solvents such as ethers, alkanes, chlorinated hydrocarbons, preferably in dichloromethane, chloroform or 1, 1, 1-trichloroethane.
- ammonium salts are prepared by reacting the carboxylic acid esters or carboxamides containing an amino group with an acid or with alkyl halides.
- the hydrochlorides are thus obtained at temperatures from -10 to 40 ° C., preferably at 0 to 20 ° C., in inert solvents, preferably in hexane, by introducing gaseous HC1.
- the compounds according to the invention are preferably stored under an inert atmosphere and in the absence of light at temperatures from -30 to 4 ° C.
- the new compounds are suitable for the treatment of acute and chronic inflammatory processes, rheumatic diseases, thrombosis and infarcts, psoriasis, shock lung and septic shock. They also have a protective effect in graft versus host disease.
- the compounds according to the invention can be administered orally or parenterally (subcutaneously, intravenously, intramuscularly, intraperitoneally).
- the dosage depends on the age, condition and weight of the patient and on the type of application.
- the daily dose of active substance is between approximately 10 and 1000 mg / kg body weight when administered orally and between approximately 1 and 100 mg / kg body weight when administered parenterally.
- the new compounds can be used in the customary pharmaceutical application forms in solid or liquid form, for example as tablets, film tablets, capsules, powders, granules, dragées, suppositories, solutions, ointments, creams or sprays. These are manufactured in the usual way.
- the active ingredients can be processed with the usual pharmaceutical auxiliaries such as tablet binders, fillers, preservatives, tablet disintegrants, flow regulators, plasticizers, wetting agents, dispersants, emulsifiers, solvents, retardants, antioxidants and / or propellants (cf. H. Sucker et al:
- the application forms obtained in this way normally contain the active ingredient in an amount of 0.1 to 99% by weight.
- AAV 1 Production of the carboxylic acid chlorides:
- AAV 2 Preparation of the carboxylic acid esters and carboxamides:
- Gaseous HC1 is introduced into a solution of amino compound in hexane (5 to 25 ml / mmol amine) under N2 atmosphere at 20 ° C for 30 min. The solvent is then distilled off under reduced pressure at 40 to 50 ° C.
- 3.0 mmol eicosapentaenoic acid chloride were prepared from 0.92 g (3.0 mmol) EPA and 1.0 g (7.9 mmol) oxalic acid dichloride in 10 ml chloroform according to AAV 1 and with 0.4 g (4.5 mmol) 2-Dimethylaminoethanol and 0.25 g (2.0 mmol) of 4-dimethylaminopyridine in 10 ml of dichloromethane according to AAV 2 (reaction time 14 h). The product was purified by chromatography (MTB / Hex / EtOH 1: 1: 1). The yield was 0.86 g (76%).
- 16.6 mmol eicosapentaenoic acid chloride were prepared from 5.0 g (16.6 mmol) EPA and 15.0 g (118.1 mmol) oxalic acid dichloride in 75 ml chloroform according to AAV 1 and 1.94 g (16.6 mmol) 2-diethyl-a inoethanol and 2.03 g (16.6 mmol) 4-dimethylaminopyridine in 70 ml dichloromethane according to AAV 2 (reaction time 18 h). The product was purified by chromatography (MTB / Hex / EtOH 1: 1: 1). The yield was 3.24 g (49%).
- 16.6 mmol eicosapentaenoic acid chloride were prepared from 5.0 g (16.6 mmol) EPA and 15.0 g (118.1 mmol) oxalic acid dichloride in 75 ml chloroform according to AAV 1 and with 1.71 g (16.6 mmol) of 3-dimethylaminopropanol and 2.03 g (16.6 mmol) of 4-dimethylaminopyridine in 70 ml of dichloromethane according to AAV 2 (reaction time 18 h). The product was purified by chromatography (MTB, MTB / EtOH 1: 1). The yield was 1.99 g (31%).
- 16.6 mmol of eicosapentaenoic acid chloride were prepared from 5.0 g (16.6 mmol) of EPA and 15.0 g (118.1 mmol) of oxalic acid dichloride in 75 ml of chloroform according to AAV 1 and with 1.54 g (14.9 mmol) of 1-dimethylamino-2-propanol and 2.03 g (16.6 mmol) of 4-dimethylamino-pyridine in 70 ml of dichloromethane according to AAV 2 (reaction time 18 h). The product was purified by chromatography (MTB, MTB / EtOH 1: 1). The yield was 2.44 g (38%).
- 16.6 mmol eicosapentaenoic acid chloride were prepared from 5.0 g (16.6 mmol) EPA and 15.0 g (118.1 mmol) oxalic acid dichloride in 75 ml chloroform according to AAV 1 and with 1.32 g (14.9 mmol) N, N-dimethylethylenediamine and 2.03 g (16.6 mmol) 4-dimethylaminopyridine in 70 ml dichloromethane according to AAV 2 (reaction time 18 h). The product was purified chromatographically (MTB / Hex / EtOH 1: 1: 1). The yield was 1.31 g (24%).
- 10.8 mmol of octadecatrienoic acid chloride were prepared from 3.0 g (10.8 mmol) of octadecatrienoic acid and 3.40 g (26.8 mmol) of oxalic acid dichloride in 45 ml of chloroform according to AAV 1 and with 0.97 g (10.8 mmol) 2-Dimethylaminoethanol and 1.33 g (10.8 mmol) 4-dimethylaminopyridine in 30 ml dichloromethane according to AAV 2 (reaction time 18 h). The product was purified by chromatography (MTB / Hex / EtOH 1: 1: 1). The yield was 1.65 g (44%).
- 16.6 mmol of eicosapentaenoic acid chloride were prepared from 5.0 g (16.6 mmol) of EPA and 15.0 g (118.1 mmol) of oxalic acid dichloride in 75 ml of chloroform according to AAV 1 and with 2.65 g (16.6 mmol) of N-tert-butyloxycarbonyl-2-aminoethanol and 1.5 g (12.3 mmol) of 4-dimethyl-a-inopyridine in 50 ml of dichloromethane according to AAV 2 (reaction time 4 h). The product was purified by chromatography (MTB / Hex / EtOH 1: 1: 2). The yield was 7.04 g (92%).
- Example 6 0.3 g (0.75 mmol) of the substance of Example 6 were reacted in 20 ml of hexane according to AAV 3 with gaseous HC1. The yield was 0.31 g (95%).
- Example 7 0.7 g (2.0 mmol) of the substance of Example 7 were reacted in 10 ml of hexane according to AAV 3 with gaseous HC1. The yield was 0.77 g (100%).
- Stimulated granulocytes are characterized by the release of a large number of lytic enzymes and, in particular, large amounts of various oxygen radicals.
- the inhibitory effect on neutrophil granulocytes activated with various stimuli was first measured in a chemiluminescence assay.
- the chemiluminescence technique is a very sensitive detection system for oxygen radicals.
- polymorphonuclear neutrophils from heparinized whole blood from healthy donors were subjected to dextran sedimentation (Dextran T250, 3%; 1 xg; 1 h at room temperature) and subsequent separation using a Percoll two-stage gradient ( 60% / 68% PERCOLL in HBSS without Ca z + / Mg z + ) at 600 xg over 30 min at 4 ° C.
- the cell fraction from the 60% / 68% PERCOLL intermediate phase was washed twice and consisted of> 98% neutrophil granulocytes.
- the chiluminescence measurements [Meth. Enzyme.
- Table 1 shows that the new substances at 5 min. Pre-incubation has a good inhibitory effect on the activation of human polymorphonuclear neutrophils.
- Examples 1 and 8 show that the ester form has a significantly better inhibitory effect than the ester components alone or as an equimolar mixture of the acid and the amino alcohol component.
- the reference substance pentoxifylline also results in lower inhibitory effects.
- Example 8 shows that the substance of Example 8 has a high potency against other natural stimuli (PAF / fMLP) of PMN activation, but not against the phorbol ester and PKC activator PMA. Table 2
- Table 3 shows the superiority of Example 8 over the tri-EPA glycerol in terms of rapid development of the inhibitory effect in human PMN.
- the rapid effect on the radical release is neither due to any radical scavenger properties (control in the cell-free system negative) nor to a cytotoxic effect of the test substance on the effector cells.
- the viability after 4 h of pre-incubation and 30 min exposure to TNF was always> 90%.
- Splenocytes from Balb / c mice were used to evaluate the effect of omega-3 fatty acids and their derivatives on lymphoproliferation. Stimulation of the splenocyte culture with LPS leads to the proliferation of the B cell subpopulation, stimulation with Enterotoxin B to the T cell proliferation.
- the splenocyte cultures were preincubated with different concentrations of the test substances in microtiter plates for 2 hours and then induced with LPS or enterotoxin B for proliferation.
- the relative growth inhibition, based on the corresponding control without inhibitor, was calculated from the count yield of the incorporated [3H] -thymidine after two days of incubation at 37 ° C. and then a 6-hour 3H-tymidine pulse (1 ⁇ Ci / 200 ⁇ l / well) and then determined the half-maximum inhibitory concentrations (IC50).
- Table 4 shows for eicosapentaenoic acid and for the esters of Examples 1 and 8 an equally pronounced antiproliferative effect on both the B and T cell subpopulation of mouse splenocytes
- mice The sensitivity of mice to lipopolysaccharide depends on the strain and age of the animals. Preliminary experiments with Balb / c mice gave a lethal dose of 20 mg / kg IV. in 6-8 week old animals.
- test substances in endotoxin shock was investigated by i.v. the inhibitors 30 min before LPS administration (20 mg / kg). were applied.
- Table 5 shows the protective effects of the substances in comparison with eicosapentaenoic acid and eicosapentaenoic acid triglyceride.
- the animals (Balb / c mice, 6-8 weeks old) were treated once with different doses of the sample substances (bolus, iv). 30 min later, an iv application with 20 mg / kg lipopolysaccharide (LPS) from E. coli, serotype 0 111 B4 was carried out. This dose was found to be 100% lethal in the control group.
- LPS lipopolysaccharide
- 5-10 animals were tested in at least 2 independent experiments.
- the table shows the percentage survival rate for the specified dose range.
- Example 7 80-100 20
- Example 8 80-100 5-20
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Abstract
Described are compounds of the formula: R?1-X-Y-NR2R3¿, plus their salts with physiologically tolerable acids and their C¿1-2?-trialkylammonium salts. These compounds are suitable for use in the treatment of illnesses.
Description
Omega-3 mehrfach ungesättigte Fettsäurederivate, ihre Herstellung und VerwendungOmega-3 polyunsaturated fatty acid derivatives, their production and use
Beschreibungdescription
Die vorliegende Erfindung betrifft neue omega-3 mehrfach unge¬ sättigte Fettsäurederivate, deren Herstellung und ihre Verwendung zur Behandlung von Entzündungsprozessen und von septischem Schock.The present invention relates to new omega-3 polyunsaturated fatty acid derivatives, their preparation and their use for the treatment of inflammatory processes and septic shock.
Eicosapentaensäure (EPA) und Docosahexaensäure (DHA), die in der Natur in relativ hohen Konzentrationen in Fischölen auftreten, sind für ihre antiinflammatorischen Eigenschaften bekannt [Acta. Med. Scand. 208, 401 (1980); J. Clin. Immunol . tS, 402 (1986)]. Inzwischen wurde erkannt, daß einer der Wirkungsmechanismen dieser Fettsäuren auf den Einfluß auf die Arachidonsäure-Kaskade zurückzuführen ist. So führt z.B. eine EPA-Diät zu einer ver¬ mehrten Bildung des Intermediärproduktes Leukotrien B5 bei gleichzeitiger Verminderung von Leukotrien B4. letzteres besitzt ein wesentlich höheres inflammatorisches Potential als Leuko¬ trien B5 [J. Clin. Invest. j"4 1922 (1984); J. Biol. Chem. 259, 7615 (1984)]; ebenso ist die endogene Bildung des platelet activating factor reduziert [Biochem. Biophys. Acta 922, 214 (1987)].Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which occur in nature in relatively high concentrations in fish oils, are known for their anti-inflammatory properties [Acta. Med. Scand. 208, 401 (1980); J. Clin. Immunol. tS, 402 (1986)]. In the meantime, it has been recognized that one of the mechanisms of action of these fatty acids is due to the influence on the arachidonic acid cascade. For example, an EPA diet leads to an increased formation of the intermediate product leukotriene B5 with a simultaneous reduction in leukotriene B4. the latter has a significantly higher inflammatory potential than leukotriene B 5 [J. Clin. Invest. j " 4 1922 (1984); J. Biol. Chem. 259, 7615 (1984)]; likewise the endogenous formation of the platelet activating factor is reduced [Biochem. Biophys. Acta 922, 214 (1987)].
Eine Reihe von anderen Funktionen wird durch EPA und DHA beein¬ flußt. In Tieren und menschlichen Probanden wurde die Hemmung der Freisetzung von Mediatoren aus Monozyten (Tumornekrosefaktor, Interleukin 1) nach hinreichend langer Diät beschrieben [N. Engl. J. Med. 320, 265 (1989)]; ebenso wird die Thrombozyten-Aggrega- tion durch EPA gehemmt [Nephron 43, 196 (1986)]. Auf die Prolife¬ ration von Ly phozyten wirkt EPA dämpfend; darüberhinaus in¬ hibiert es die Freisetzung von lytischen Enzymen, Sauerstoff¬ radikalen und die Adhärenz von Granulozyten an das Endothelium [N. Engl. J. Med. 312, 1217 (1985); Biochem. Biophys. Res. Comm. 137, 10094 (1986)].A number of other functions are influenced by EPA and DHA. The inhibition of the release of mediators from monocytes (tumor necrosis factor, interleukin 1) was described in animals and human subjects after a sufficiently long diet [N. Engl. J. Med. 320, 265 (1989)]; platelet aggregation is also inhibited by EPA [Nephron 43, 196 (1986)]. EPA has a dampening effect on the proliferation of lymphocytes; moreover, it inhibits the release of lytic enzymes, oxygen radicals and the adherence of granulocytes to the endothelium [N. Engl. J. Med. 312, 1217 (1985); Biochem. Biophys. Res. Comm. 137, 10094 (1986)].
Die kurz umrissenen Hemmeffekte können in den verschiedensten Krankheitsbildern von therapeutischem Nutzen sein; insbesondere
bei akuten und chronischen Entzündungsprozessen [J. Immunol. 134, 1914 (1985)], rheumatoiden Erkrankungen [J. Rheumatol. 1_5_, 1471 (1988)], Thrombosen und Infarkt, Psoriasis [Lancet Vol.l, 378 (1988)] und septischem Schock. Ebenso ist eine protektive Wirkung bei Graft versus Host Disease zu erwarten. Im Falle des septischen Schocks muß der Fettsäureester intravenös appliziert werden, um einen schnellen Schutzeffekt zu erreichen. Kürzlich wurde gezeigt, daß eine wirksame Reduktion von LTB4 bei Verwen¬ dung von Eicosapentaensäuretriclycerid bereits 6 Stunden nach i.v. Gabe erreichbar ist [Abstract 7th Intern. Conf. onThe briefly outlined inhibitory effects can be of therapeutic use in a wide variety of clinical pictures; in particular in acute and chronic inflammatory processes [J. Immunol. 134, 1914 (1985)], rheumatoid diseases [J. Rheumatol. 1_5_, 1471 (1988)], thrombosis and infarction, psoriasis [Lancet Vol.l, 378 (1988)] and septic shock. A protective effect in graft versus host disease is also expected. In the case of septic shock, the fatty acid ester must be administered intravenously in order to achieve a quick protective effect. It has recently been shown that an effective reduction of LTB4 can be achieved as early as 6 hours after iv administration when eicosapentaenoic acid triclyceride is used [Abstract 7th Intern. Conf. on
Prostaglandis and Related Compounds, Florence 1990, S. 47].Prostaglandis and Related Compounds, Florence 1990, p. 47].
Es wurden nun Verbindungen mit besseren Eigenschaften gefunden.Compounds with better properties have now been found.
Gegenstand der Erfindung sind Verbindungen der Formel IThe invention relates to compounds of the formula I.
Rl-X-Y- NR2R3 I,Rl-X-Y- NR2R3 I,
worinwherein
Ri der Acylrest von 5,8, 11, 14, 17-Eicosapentaensäure,Ri is the acyl residue of 5,8, 11, 14, 17-eicosapentaenoic acid,
4,7,10, 13,16, 19-Docosahexaensäure oder 9, 12, 15-0ctadeca- triensäure,4,7,10, 13,16, 19-docosahexaenoic acid or 9, 12, 15-0ctadecatrienoic acid,
X ein Sauerstoffatom oder ein NH-Gruppe,X is an oxygen atom or an NH group,
Y eine C2-3-Alkylengruppe,Y is a C2-C3 alkylene group,
R2 und R3 Wasserstoffatome oder Cι_2-AlkylgruppenR2 and R3 are hydrogen atoms or Cι_2 alkyl groups
darstellen, sowie deren Salze mit physiologisch verträglichen Säuren und Cι_2-Trialkylammoniumsalze.
represent, and their salts with physiologically acceptable acids and Cι_2-trialkylammonium salts.
5, 8, 11 , 14, 17-Ei cosapentaensäure (= EPA) hat die Formel5, 8, 11, 14, 17-egg cosapentaenoic acid (= EPA) has the formula
-COOH,-COOH,
4, 7, 10, 13, 16, 19-Docosahexaensäure (= DHA ) di e Formel OOH und4, 7, 10, 13, 16, 19-docosahexaenoic acid (= DHA) the formula OOH and
9, 12, 15-Octadecatriensäure (= o-Linolensäure) die Formel COOH.9, 12, 15-octadecatrienoic acid (= o-linolenic acid) the formula COOH.
In der Formel I ist von den Säureresten der der Eicosapentaen- säure bevorzugt. X ist vorzugsweise ein Sauerstoffatom, Y ist vorzugsweise eine C2-Alkylengruppe und R2 und R3 sind vorzugs¬ weise Methyl- oder Ethylreste, inbesondere aber Methylreste. Vorzugsweise liegen die Verbindungen als Salze mit physiologisch verträglichen Säuren vor.Of the acid residues in formula I, that of eicosapentaenoic acid is preferred. X is preferably an oxygen atom, Y is preferably a C2-alkylene group and R2 and R3 are preferably methyl or ethyl radicals, but especially methyl radicals. The compounds are preferably in the form of salts with physiologically tolerable acids.
Als physiologisch verträgliche Säuren sind insbesondere zu nennen: Salzsäure, Zitronensäure, Weinsäure, Milchsäure, Phos¬ phorsäure, Methansulfonsäure, Essigsäure, Ameisensäure, Malein¬ säure, Fumarsäure, Äpfelsäure, Bernsteinsäure, Malonsäure, Schwefelsäure, L-Glutaminsäure, L-Asparaginsäure, Brenztrauben- säure, Schleimsäure, Benzoesäure, Glucuronsäure, Oxalsäure,In particular, the following can be mentioned as physiologically acceptable acids: hydrochloric acid, citric acid, tartaric acid, lactic acid, phosphoric acid, methanesulfonic acid, acetic acid, formic acid, maleic acid, fumaric acid, malic acid, succinic acid, malonic acid, sulfuric acid, L-glutamic acid, L-aspartic acid, pyruvic acid - acid, mucic acid, benzoic acid, glucuronic acid, oxalic acid,
Ascorbinsäure, Acetylglycin, Orotsäure. Bevorzugte Salze sind die Hydrochloride und die Trialkylaπunoniumchloride.Ascorbic acid, acetylglycine, orotic acid. Preferred salts are the hydrochlorides and the trialkylaunonium chlorides.
Die erfindungsgemäßen Verbindungen lassen sich nach allgemeinen Methoden zur Synthese von Carbonsäureestern bzw. Carbonsäure- amiden und gegebenenfalls anschließender Ammoniumsalzbildung herstellen.The compounds according to the invention can be prepared by general methods for the synthesis of carboxylic acid esters or carboxylic acid amides and, if appropriate, subsequent ammonium salt formation.
Zur Synthese der Carbonsäureester bzw. Carbonsäureamide sind vorzugsweise die Umsetzung der Carbonsäurechloride mit den ent¬ sprechenden Alkoholen bzw. Aminen in Gegenwart tertiärer Amin- basen, z.B. 4-Dimethylaminopyridin, oder die Umsetzung der Carbonsäuren mit den entsprechenden Alkoholen bzw. Aminen in Gegenwart wasserentziehender Mittel, z.B. Dicyclohexylcarbodi imid mit 4-Dimethylaminopyridin als Aktivator, geeignet.
Die Knüpfung der Ester- bzw. Amidbindungen geschieht bei Tempe¬ raturen von -10 bis 100°C, vorzugsweise bei Temperaturen von 0 bis 40°C in inerten Lösungsmitteln wie Ethern, Alkanen, chlorier¬ ten Kohlenwasserstoffen, vorzugsweise in Dichlormethan, Chloro- form oder 1, 1, 1-Trichlorethan.The synthesis of the carboxylic acid esters or carboxamides is preferably the reaction of the carboxylic acid chlorides with the corresponding alcohols or amines in the presence of tertiary amine bases, for example 4-dimethylaminopyridine, or the reaction of the carboxylic acids with the corresponding alcohols or amines in the presence of dehydrating agents , for example dicyclohexylcarbodimide with 4-dimethylaminopyridine as an activator. The ester or amide bonds are formed at temperatures from -10 to 100 ° C., preferably at temperatures from 0 to 40 ° C. in inert solvents such as ethers, alkanes, chlorinated hydrocarbons, preferably in dichloromethane, chloroform or 1, 1, 1-trichloroethane.
Die Ammoniu salze werden durch Umsetzung der eine A inogruppe enthaltenden Carbonsäureester bzw. Carbonsäureamide mit einer Säure bzw. mit Alkylhalogeniden hergestellt.The ammonium salts are prepared by reacting the carboxylic acid esters or carboxamides containing an amino group with an acid or with alkyl halides.
So erhält man die Hydrochloride bei Temperaturen von -10 bis 40°C, vorzugsweise bei 0 bis 20°C, in inerten Lösungsmitteln, vorzugsweise in Hexan, durch Einleiten von gasförmigem HC1.The hydrochlorides are thus obtained at temperatures from -10 to 40 ° C., preferably at 0 to 20 ° C., in inert solvents, preferably in hexane, by introducing gaseous HC1.
Die erschöpfend alkylierten Ammoniumverbindungen werden beiThe exhaustively alkylated ammonium compounds are at
Temperaturen von 0 bis 100°C, vorzugsweise bei Temperaturen von 20 bis 60°C, in inerten Lösungsmitteln, vorzugsweise in polaren Lösungsmitteln wie Nitro ethan, durch Umsetzung der Carbonsäure¬ ester bzw. Carbonsäureamide mit C__2-Alkylhalogeniden, vorzugs- weise mit Methylchlorid, hergestellt.Temperatures from 0 to 100 ° C, preferably at temperatures from 20 to 60 ° C, in inert solvents, preferably in polar solvents such as nitroethane, by reacting the carboxylic acid esters or carboxamides with C 2 alkyl halides, preferably with methyl chloride, manufactured.
Die erfindungsgemäßen Verbindungen werden vorzugsweise unter Inertatmosphäre und Lichtausschluß bei Temperaturen von -30 bis 4°C aufbewahrt.The compounds according to the invention are preferably stored under an inert atmosphere and in the absence of light at temperatures from -30 to 4 ° C.
Die neuen Verbindungen eignen sich zur Behandlung von akuten und chronischen Entzündungsprozessen, rheumatische Erkrankungen, Thrombose und Infarkten, Psoriasis, Schocklunge und septischem Schock. Weiter zeigen sie eine protektive Wirkung bei Graft versus Host Disease.The new compounds are suitable for the treatment of acute and chronic inflammatory processes, rheumatic diseases, thrombosis and infarcts, psoriasis, shock lung and septic shock. They also have a protective effect in graft versus host disease.
Die erfindungsgemäßen Verbindungen können in üblicher Weise je nach Indikation oral oder parenteral (subkutan, intravenös, intramuskulär, intraperitoneal) verabfolgt werden.Depending on the indication, the compounds according to the invention can be administered orally or parenterally (subcutaneously, intravenously, intramuscularly, intraperitoneally).
Die Dosierung hängt vom Alter, Zustand und Gewicht des Patienten sowie von der Applikationsart ab. In der Regel beträgt die täg¬ liche Wirkstoffdosis zwischen etwa 10 und 1000 mg/kg Körper¬ gewicht bei oraler Gabe und zwischen etwa 1 und 100 mg/kg Körper- gewicht bei parenteraler Gabe.
Die neuen Verbindungen können in den gebräuchlichen galenischen Applikationsformen fest oder flüssig angewendet werden, z.B. als Tabletten, Filmtabletten, Kapseln, Pulver, Granulate, Dragees, Suppositorien, Lösungen, Salben, Cremes oder Sprays. Diese werden in üblicher Weise hergestellt. Die Wirkstoffe können dabei mit den üblichen galenischen Hilfsmitteln wie Tablettenbindern, Füll¬ stoffen, Konservierungsmitteln, Tablettensprengmitteln, Flie߬ reguliermitteln, Weichmachern, Netzmitteln, Dispergiermitteln, Emulgatoren, Lösungsmitteln, Retardierungsmitteln, Antioxidantien und/oder Treibgasen verarbeitet werden (vgl. H. Sucker et al :The dosage depends on the age, condition and weight of the patient and on the type of application. As a rule, the daily dose of active substance is between approximately 10 and 1000 mg / kg body weight when administered orally and between approximately 1 and 100 mg / kg body weight when administered parenterally. The new compounds can be used in the customary pharmaceutical application forms in solid or liquid form, for example as tablets, film tablets, capsules, powders, granules, dragées, suppositories, solutions, ointments, creams or sprays. These are manufactured in the usual way. The active ingredients can be processed with the usual pharmaceutical auxiliaries such as tablet binders, fillers, preservatives, tablet disintegrants, flow regulators, plasticizers, wetting agents, dispersants, emulsifiers, solvents, retardants, antioxidants and / or propellants (cf. H. Sucker et al:
Pharmazeutische Technologie, Thie e-Verlag, Stuttgart, 1978). Die so erhaltenen Applikationsformen enthalten den Wirstoff normaler¬ weise in einer Menge von 0,1 bis 99 Gew.-%.Pharmaceutical Technology, Thie e-Verlag, Stuttgart, 1978). The application forms obtained in this way normally contain the active ingredient in an amount of 0.1 to 99% by weight.
Die im folgenden aufgeführten Beispiele beschrieben die Erfindung detailliert, schränken die Erfindung aber in keiner Weise ein.The examples listed below describe the invention in detail, but in no way limit the invention.
HerstellVorschriften:Manufacturing regulations:
Allgemeine Arbeitsvorschriften (AAV) :General labor regulations (AAV):
AAV 1: Herstellung der Carbonsäurechloride:AAV 1: Production of the carboxylic acid chlorides:
1 Äquivalent Carbonsäure wird in absolutem Chloroform oder 1,1,1- Trichlorethan vorgelegt (3 bis 5 ml/mmol Carbonsäure) und unter N2~Atmosphäre bei 20°C vorsichtig mit 2 bis 10 Äquivalenten Oxal¬ säuredichlorid versetzt. Nach 2 h bei 20°C werden Lösungsmittel und überschüssiges Oxalsäuredichlorid unter reduziertem Druck bei 40°C abdestilliert. Das Säurechlorid wird in die weiteren Umset- Zungen roh eingesetzt.1 equivalent of carboxylic acid is placed in absolute chloroform or 1,1,1-trichloroethane (3 to 5 ml / mmol of carboxylic acid) and 2 to 10 equivalents of oxalic acid dichloride are carefully added under an N 2 atmosphere at 20 ° C. After 2 h at 20 ° C., solvent and excess oxalic acid dichloride are distilled off at 40 ° C. under reduced pressure. The acid chloride is used raw in the further reaction tongues.
AAV 2: Herstellung der Carbonsäureester und Carbonsäureamide:AAV 2: Preparation of the carboxylic acid esters and carboxamides:
0,65 bis 1,5 Äquivalente Alkohol bzw. Amin und 0,75 bis 1,1 Äqui- valente Base (4-Dimethylaminopyridin) werden in absolutem Di- chlormethan, Chloroform oder 1, 1, 1-Trichlorethan (3 bis 5 ml/mmol Carbonsäurechlorid) unter N2~Atmosphäre bei 20°C vorsichtig mit 1 Äquivalent in wenig Lösungsmittel gelöstem Carbonsäurechlorid umgesetzt. Nach beendeter Reaktion (DC-Kontrolle) wird die Reak- tionsmischung unter reduziertem Druck bei 40°C eingeengt und das Produkt chromatographisch über Kieselgel 60 gereinigt.
AAV 3: Herstellung der Hydrochloride:0.65 to 1.5 equivalents of alcohol or amine and 0.75 to 1.1 equivalents of base (4-dimethylaminopyridine) are dissolved in absolute dichloromethane, chloroform or 1, 1, 1-trichloroethane (3 to 5 ml / mmol carboxylic acid chloride) under N2 ~ atmosphere at 20 ° C carefully reacted with 1 equivalent of carboxylic acid chloride dissolved in a little solvent. When the reaction has ended (TLC control), the reaction mixture is concentrated under reduced pressure at 40 ° C. and the product is purified by chromatography on silica gel 60. AAV 3: Production of the hydrochloride:
In eine Lösung von Aminoverbindung in Hexan (5 bis 25 ml/mmol Amin) wird unter N2~Atmosphäre bei 20°C 30 min HC1 gasfärmig ein- geleitet. Anschliessend wird das Lösungsmittel unter reduziertem Druck bei 40 bis 50°C abdestilliert.Gaseous HC1 is introduced into a solution of amino compound in hexane (5 to 25 ml / mmol amine) under N2 atmosphere at 20 ° C for 30 min. The solvent is then distilled off under reduced pressure at 40 to 50 ° C.
Spezielle ArbeitsvorschriftenSpecial working regulations
Beispiel 1example 1
Eicosapentaensäure-(2-dimethylaminoethyl)-esterEicosapentaenoic acid (2-dimethylaminoethyl) ester
3,0 mmol Eicosapentaensäurechlorid wurden aus 0,92 g (3,0 mmol) EPA und 1,0 g (7,9 mmol) Oxalsäuredichlorid in 10 ml Chloroform nach AAV 1 hergestellt und mit 0,4 g (4,5 mmol) 2-Dimethylamino- ethanol und 0,25 g (2,0 mmol) 4-Dimethylaminopyridin in 10 ml Dichlormethan nach AAV 2 umgesetzt (Reaktionszeit 14 h). Das Produkt wurde chromatographisch gereinigt (MTB/Hex/EtOH 1:1:1). Die Ausbeute betrug 0,86 g (76 %) .3.0 mmol eicosapentaenoic acid chloride were prepared from 0.92 g (3.0 mmol) EPA and 1.0 g (7.9 mmol) oxalic acid dichloride in 10 ml chloroform according to AAV 1 and with 0.4 g (4.5 mmol) 2-Dimethylaminoethanol and 0.25 g (2.0 mmol) of 4-dimethylaminopyridine in 10 ml of dichloromethane according to AAV 2 (reaction time 14 h). The product was purified by chromatography (MTB / Hex / EtOH 1: 1: 1). The yield was 0.86 g (76%).
1H-NMR (270 MHz, CDCl3), δ (pp ) : 0.98 (t,J=8HZ,3H) ; 1.70 (m,2H) ; 2.05-2.15(kB,4H); 2.25(s,6H); 2.35(t,J=8Hz,2H) ;2.60(t, >=5,5Hz, 2H); 2.80-3.00(kB,8H) ; 4.20(t,J=5,5HZ,2H) ; 5.20-5.50(kB, 10H) .1H NMR (270 MHz, CDCl 3 ), δ (pp): 0.98 (t, J = 8HZ, 3H); 1.70 (m, 2H); 2.05-2.15 (kB, 4H); 2.25 (s, 6H); 2.35 (t, J = 8Hz, 2H); 2.60 (t,> = 5.5Hz, 2H); 2.80-3.00 (kB, 8H); 4.20 (t, J = 5.5HZ, 2H); 5.20-5.50 (kB, 10H).
Beispiel 2Example 2
Eicosapentaensäure-(2-dieth laminoethy1)-esterEicosapentaenoic acid (2-dieth laminoethy1) ester
16,6 mmol Eicosapentaensäurechlorid wurden aus 5,0 g (16,6 mmol) EPA und 15,0 g (118,1 mmol) Oxalsäuredichlorid in 75 ml Chloro¬ form nach AAV 1 hergestellt und mit 1,94 g (16,6 mmol) 2-Diethyl- a inoethanol und 2,03 g (16,6 mmol) 4-Dimethylaminopyridin in 70 ml Dichlormethan nach AAV 2 umgesetzt (Reaktionszeit 18 h) .
Das Produkt wurde chromatographisch gereinigt (MTB/Hex/EtOH 1:1:1). Die Ausbeute betrug 3,24 g (49 %) . 16.6 mmol eicosapentaenoic acid chloride were prepared from 5.0 g (16.6 mmol) EPA and 15.0 g (118.1 mmol) oxalic acid dichloride in 75 ml chloroform according to AAV 1 and 1.94 g (16.6 mmol) 2-diethyl-a inoethanol and 2.03 g (16.6 mmol) 4-dimethylaminopyridine in 70 ml dichloromethane according to AAV 2 (reaction time 18 h). The product was purified by chromatography (MTB / Hex / EtOH 1: 1: 1). The yield was 3.24 g (49%).
1H-NMR (360 MHZ, CDC13), . (ppm) : 0.98(t, J=8Hz, 3H) ; 1.05(t, =8HZ, 6H) ; 1.70(m,2H); 2.05-2.15(kB, 4H) ; 2.35(t, J=8Hz, 2H) ; 2.58(q, J=8HZ,4H); 2.70(t,J=6Hz,2H); 2.75-2.90(kB, 8H) ; 4.15(t, J=6Hz, 2H) ; 5.30-5.45(kB, 10H).1H-NMR (360 MHz, CDC1 3 ),. (ppm): 0.98 (t, J = 8Hz, 3H); 1.05 (t, = 8HZ, 6H); 1.70 (m, 2H); 2.05-2.15 (kB, 4H); 2.35 (t, J = 8Hz, 2H); 2.58 (q, J = 8HZ, 4H); 2.70 (t, J = 6Hz, 2H); 2.75-2.90 (kB, 8H); 4.15 (t, J = 6Hz, 2H); 5.30-5.45 (kB, 10H).
Beispiel 3Example 3
Eicosapentaensäure-(3-dimethylaminopropyl)-esterEicosapentaenoic acid (3-dimethylaminopropyl) ester
16,6 mmol Eicosapentaensäurechlorid wurden aus 5,0 g (16,6 mmol) EPA und 15,0 g (118,1 mmol) Oxalsäuredichlorid in 75 ml Chloro¬ form nach AAV 1 hergestellt und mit 1,71 g (16,6 mmol) 3-Di- methylaminopropanol und 2,03 g (16,6 mmol) 4-Dimethylaminopyridin in 70 ml Dichlormethan nach AAV 2 umgesetzt (Reaktionszeit 18 h). Das Produkt wurde chromatographisch gereinigt (MTB,MTB/EtOH 1:1). Die Ausbeute betrug 1,99 g (31 %) .16.6 mmol eicosapentaenoic acid chloride were prepared from 5.0 g (16.6 mmol) EPA and 15.0 g (118.1 mmol) oxalic acid dichloride in 75 ml chloroform according to AAV 1 and with 1.71 g (16.6 mmol) of 3-dimethylaminopropanol and 2.03 g (16.6 mmol) of 4-dimethylaminopyridine in 70 ml of dichloromethane according to AAV 2 (reaction time 18 h). The product was purified by chromatography (MTB, MTB / EtOH 1: 1). The yield was 1.99 g (31%).
1H-NMR (250 MHz, CDCI3), δ (ppm): 0.98(t, =8Hz, 3H) ; 1.65-1.85(kB, 4H); 2.05-2.20(kB,4H); 2.20(s,6H); 2.25-2.40(kB,4H) ; 2.80-2.90 (kB,8H); 4.10(t,J=6Hz,2H); 5.25-5.45(kB, 10) .1H-NMR (250 MHz, CDCI3), δ (ppm): 0.98 (t, = 8Hz, 3H); 1.65-1.85 (kB, 4H); 2.05-2.20 (kB, 4H); 2.20 (s, 6H); 2.25-2.40 (kB, 4H); 2.80-2.90 (kB, 8H); 4.10 (t, J = 6Hz, 2H); 5.25-5.45 (kB, 10).
Beispiel 4Example 4
Eicosapentaensäure-(l-methyl-2-dimethylaminoethyl)esterEicosapentaenoic acid (l-methyl-2-dimethylaminoethyl) ester
16,6 mmol Eicosapentaensäurechlorid wurden aus 5,0 g (16,6 mmol) EPA und 15,0 g (118,1 mmol) Oxalsäuredichlorid in 75 ml Chloro¬ form nach AAV 1 hergestellt und mit 1,54 g (14,9 mmol) 1-Di- methylamino-2-propanol und 2,03 g (16,6 mmol) 4-Dimethylamino- pyridin in 70 ml Dichlormethan nach AAV 2 umgesetzt (Reaktions¬ zeit 18 h). Das Produkt wurde chromatographisch gereinigt (MTB, MTB/EtOH 1:1). Die Ausbeute betrug 2,44 g (38 %) .
1H-NMR (250 MHZ, CDCl3), δ (ppm): 0.98(t, =8Hz, 3H) ; 1.20(d, =8Hz, 3H); 1.65-1.75(m,2H); 2.00-2.15(kB, 4H) ; 2.25(s,6H); 2.25-2.35 (kB,3H); 2.50(dd,J=13Hz/8Hz, 1H); 2.75-2.90(kB,8H) ; 5.05(m, 1H); 5.25-5.45(kB, 10H).16.6 mmol of eicosapentaenoic acid chloride were prepared from 5.0 g (16.6 mmol) of EPA and 15.0 g (118.1 mmol) of oxalic acid dichloride in 75 ml of chloroform according to AAV 1 and with 1.54 g (14.9 mmol) of 1-dimethylamino-2-propanol and 2.03 g (16.6 mmol) of 4-dimethylamino-pyridine in 70 ml of dichloromethane according to AAV 2 (reaction time 18 h). The product was purified by chromatography (MTB, MTB / EtOH 1: 1). The yield was 2.44 g (38%). 1H-NMR (250 MHz, CDCl 3 ), δ (ppm): 0.98 (t, = 8Hz, 3H); 1.20 (d, = 8Hz, 3H); 1.65-1.75 (m, 2H); 2.00-2.15 (kB, 4H); 2.25 (s, 6H); 2.25-2.35 (kB, 3H); 2.50 (dd, J = 13Hz / 8Hz, 1H); 2.75-2.90 (kB, 8H); 5.05 (m, 1H); 5.25-5.45 (kB, 10H).
Beispiel 5Example 5
Ei cosapentaensäure- ( 2-d imethy 1 ami noethy 1 ) -ami dEgg cosapentaenoic acid (2-d imethy 1 ami noethy 1) -ami d
16,6 mmol Eicosapentaensäurechlorid wurden aus 5,0 g (16,6 mmol) EPA und 15,0 g (118,1 mmol) Oxalsäuredichlorid in 75 ml Chloro¬ form nach AAV 1 hergestellt und mit 1,32 g (14,9 mmol) N,N-Di- methylethylendiamin und 2,03 g (16,6 mmol) 4-Dimethylaminopyridin in 70 ml Dichlormethan nach AAV 2 umgesetzt (Reaktionszeit 18 h). Das Produkt wurde Chromatographiseh gereinigt (MTB/Hex/EtOH 1:1:1). Die Ausbeute betrug 1,31 g (24 %). 16.6 mmol eicosapentaenoic acid chloride were prepared from 5.0 g (16.6 mmol) EPA and 15.0 g (118.1 mmol) oxalic acid dichloride in 75 ml chloroform according to AAV 1 and with 1.32 g (14.9 mmol) N, N-dimethylethylenediamine and 2.03 g (16.6 mmol) 4-dimethylaminopyridine in 70 ml dichloromethane according to AAV 2 (reaction time 18 h). The product was purified chromatographically (MTB / Hex / EtOH 1: 1: 1). The yield was 1.31 g (24%).
1H-NMR (300 MHz, CDC13), _ (ppm): 0.98(t, =8Hz, 3H) ; 1.70(m,2H); 2.00-2.25(kB,6H); 2.20(s,6H); 2.40(t,0=6Hz,2H) ; 2.75-2.90(kB, 8H) ; 3.32(m,2H); 5.30-5.50(kB, 10H) ; 6.00(s,lH).1H-NMR (300 MHz, CDC1 3 ), _ (ppm): 0.98 (t, = 8Hz, 3H); 1.70 (m, 2H); 2.00-2.25 (kB, 6H); 2.20 (s, 6H); 2.40 (t, 0 = 6Hz, 2H); 2.75-2.90 (kB, 8H); 3.32 (m, 2H); 5.30-5.50 (kB, 10H); 6.00 (s, lH).
Beispiel 6Example 6
Docosahexaensäure-(2-dimethy1aminoeth l)-esterDocosahexaenoic acid (2-dimethylaminoeth l) ester
9,2 mmol Docosahexaensäurechlorid wurden aus 3,0 g (9,2 mmol) DHA und 2,92 g (23,0 mmol) Oxalsäuredichlorid in 45 ml 1,1,1-Tri- chlorethan nach AAV 1 hergestellt und mit 0,82 g (9,2 mmol) 2-Dimethylaminoethanol und 1,10 g (9,2 mmol) 4-Dimethylamino- pyridin in 30 ml 1, 1, 1-Trichlorethan nach AAV 2 umgesetzt (Reak¬ tionszeit 18 h). Das Produkt wurde chromatographisch gereinigt (MTB/Hex/EtOH 1:1:1). Die Ausbeute betrug 1,35 g (37 %) .
IH-NMR (250 MHZ, CDCI3), δ (ppm): 0.98(t, J=8Hz, 3H) ; 2.10(m,2H) ; 2.25(s,6H); 2.35-2.40(kB, 4H) ; 2.60(t, _=6Hz, 2H) ; 2.80-2.90(kB, 10H); 4.20(t,J=6Hz,2H); 5.25-5.45(kB, 12H) .9.2 mmol of docosahexaenoyl chloride were prepared from 3.0 g (9.2 mmol) of DHA and 2.92 g (23.0 mmol) of oxalic acid dichloride in 45 ml of 1,1,1-trichloroethane according to AAV 1 and with 0, 82 g (9.2 mmol) of 2-dimethylaminoethanol and 1.10 g (9.2 mmol) of 4-dimethylamino-pyridine in 30 ml of 1,1,1-trichloroethane were converted according to AAV 2 (reaction time 18 h). The product was purified by chromatography (MTB / Hex / EtOH 1: 1: 1). The yield was 1.35 g (37%). IH-NMR (250 MHz, CDCI3), δ (ppm): 0.98 (t, J = 8Hz, 3H); 2.10 (m, 2H); 2.25 (s, 6H); 2.35-2.40 (kB, 4H); 2.60 (t, _ = 6Hz, 2H); 2.80-2.90 (kB, 10H); 4.20 (t, J = 6Hz, 2H); 5.25-5.45 (kB, 12H).
Beispiel 7Example 7
Octadecatr i ensäure- ( 2-d imethy 1 ami noethy 1 ) -esterOctadecatr ienoic acid (2-d imethy 1 ami noethy 1) ester
10,8 mmol Octadecatriensäurechlorid wurden aus 3,0 g (10,8 mmol) Octadecatriensäure und 3,40 g (26,8 mmol) Oxalsäuredichlorid in 45 ml Chloroform nach AAV 1 hergestellt und mit 0,97 g (10,8 mmol) 2-Dimethylaminoethanol und 1,33 g (10,8 mmol) 4-Di- methylaminopyridin in 30 ml Dichlormethan nach AAV 2 umgesetzt (Reaktionszeit 18 h). Das Produkt wurde chromatographisch ge- reinigt (MTB/Hex/EtOH 1:1:1). Die Ausbeute betrug 1,65 g (44 %) .10.8 mmol of octadecatrienoic acid chloride were prepared from 3.0 g (10.8 mmol) of octadecatrienoic acid and 3.40 g (26.8 mmol) of oxalic acid dichloride in 45 ml of chloroform according to AAV 1 and with 0.97 g (10.8 mmol) 2-Dimethylaminoethanol and 1.33 g (10.8 mmol) 4-dimethylaminopyridine in 30 ml dichloromethane according to AAV 2 (reaction time 18 h). The product was purified by chromatography (MTB / Hex / EtOH 1: 1: 1). The yield was 1.65 g (44%).
IH-NMR (250 MHz, CDCI3), δ (ppm): 0.95(t, J=8Hz, 3H) ; 1.15-1.35(kB, 8H); 1.55(m,2H); 1.90-2.05(kB,4H) ; 2.20(s,6H); 2.22(t, J=8HZ, 2H) ; 2.50(t,J=6Hz,2H); 2.65-2.75(kB, 4H) ; 4.10(t, =6Hz, 2H) ; 5.15-5.35 (kB,6H).IH-NMR (250 MHz, CDCI3), δ (ppm): 0.95 (t, J = 8Hz, 3H); 1.15-1.35 (kB, 8H); 1.55 (m. 2H); 1.90-2.05 (kB, 4H); 2.20 (s, 6H); 2.22 (t, J = 8HZ, 2H); 2.50 (t, J = 6Hz, 2H); 2.65-2.75 (kB, 4H); 4.10 (t, = 6Hz, 2H); 5.15-5.35 (kB, 6H).
Beispiel 8Example 8
Eicosapentaensäure-(2-dimeth 1aminoethyl)-ester-hydrochloridEicosapentaenoic acid (2-dimeth 1aminoethyl) ester hydrochloride
2,0 g (5,4 mmol) Substanz des Beispiels 1 wurden in 50 ml Hexan nach AAV 3 mit gasförmiger HC1 umgesetzt. Die Ausbeute betrug 2,16 g (98 %) .2.0 g (5.4 mmol) of substance from Example 1 were reacted in 50 ml of hexane according to AAV 3 with gaseous HC1. The yield was 2.16 g (98%).
IH-NMR (360 MHZ, CDC13), δ (ppm): 0.98(t, =9Hz, 3H) ; 1.72 (m,2H) ; 2.05-2.15 (kB,4H); 2.43(t, J=8Hz, 2H) ; 2.75-2.85(kB, ΘH) ; 2.95(d, J=8Hz,6H); 3.45(m,2H); 4.55(t, J-5, 5Hz, 2H) ; 5.30-5.45(kB, 10H) ; 11.15(m,lH).
Beispiel 9IH-NMR (360 MHz, CDC1 3 ), δ (ppm): 0.98 (t, = 9Hz, 3H); 1.72 (m, 2H); 2.05-2.15 (kB, 4H); 2.43 (t, J = 8Hz, 2H); 2.75-2.85 (kB, ΘH); 2.95 (d, J = 8Hz, 6H); 3.45 (m. 2H); 4.55 (t, J-5, 5 Hz, 2H); 5.30-5.45 (kB, 10H); 11.15 (m, lH). Example 9
Eicosapentaensäure-(2-aminoeth l)-ester-hydrochloridEicosapentaenoic acid (2-aminoeth 1) ester hydrochloride
1. N-tert.-Butyloxycarbonyl-2-aminoethanol1. N-tert-butyloxycarbonyl-2-aminoethanol
5,06 g (82,8 mmol) 2-Aminoethanol wurden mit 8,38 g (82,8 mmol) Triethyla in in 200 ml Dichlormethan vorgelegt und bei 20°C mit 18,07 g (82,8 mmol) Pyrokohlensäure-di-tert.-butylester umge- setzt. Nach 20 h wurde die Reaktionsmischung zweimal mit 100 ml ges. NaCl-Lösung und einmal mit 1 N HCl gewaschen, über NaS0 getrocknet und unter reduziertem Druck bei 60°C eingeengt. Die Ausbeute betrug 8,26 g (62 %) .5.06 g (82.8 mmol) of 2-aminoethanol were initially charged with 8.38 g (82.8 mmol) of triethyl in 200 ml of dichloromethane and at 20 ° C. with 18.07 g (82.8 mmol) of pyrocarbonic acid. di-tert-butyl ester reacted. After 20 h the reaction mixture was saturated twice with 100 ml. NaCl solution and washed once with 1N HCl, dried over NaS0 and concentrated under reduced pressure at 60 ° C. The yield was 8.26 g (62%).
2. Eicosapentaensäure-(2-tert.-butyloxycarbonyl-aminoethyl)-ester2. Eicosapentaenoic acid (2-tert-butyloxycarbonylaminoethyl) ester
16,6 mmol Eicosapentaensäurechlorid wurden aus 5,0 g (16,6 mmol) EPA und 15,0 g (118,1 mmol) Oxalsäuredichlorid in 75 ml Chloro¬ form nach AAV 1 hergestellt und mit 2,65 g (16,6 mmol) N-tert.- Butyloxycarbonyl-2-aminoethanol und 1,5 g (12,3 mmol) 4-Dimethyl- a inopyridin in 50 ml Dichlormethan nach AAV 2 umgesetzt (Reak¬ tionszeit 4 h). Das Produkt wurde chromatographisch gereinigt (MTB/Hex/EtOH 1:1:2). Die Ausbeute betrug 7,04 g (92 %).16.6 mmol of eicosapentaenoic acid chloride were prepared from 5.0 g (16.6 mmol) of EPA and 15.0 g (118.1 mmol) of oxalic acid dichloride in 75 ml of chloroform according to AAV 1 and with 2.65 g (16.6 mmol) of N-tert-butyloxycarbonyl-2-aminoethanol and 1.5 g (12.3 mmol) of 4-dimethyl-a-inopyridine in 50 ml of dichloromethane according to AAV 2 (reaction time 4 h). The product was purified by chromatography (MTB / Hex / EtOH 1: 1: 2). The yield was 7.04 g (92%).
IH-NMR (270 MHz, CDCl3), δ (ppm): 0.98(t, =8Hz,3H) ; 1.45(s,9H); 1.70(m,2H); 2.00-2.15 (kB,4H); 2.35(t,J=8Hz, 2H) ; 2.75-2.90(kB, 8H); 3.35(m,2H); 4.10(t, J=5,5Hz, 2H) ; 5.08(s,lH); 5.25-5.50 (kB,10H).IH-NMR (270 MHz, CDCl 3 ), δ (ppm): 0.98 (t, = 8Hz, 3H); 1.45 (s, 9H); 1.70 (m, 2H); 2.00-2.15 (kB, 4H); 2.35 (t, J = 8Hz, 2H); 2.75-2.90 (kB, 8H); 3.35 (m, 2H); 4.10 (t, J = 5.5Hz, 2H); 5.08 (s, 1H); 5.25-5.50 (kB, 10H).
3. Eicosapentaensäure-(2-aminoethyl)-ester-hydrochlorid3. Eicosapentaenoic acid (2-aminoethyl) ester hydrochloride
1,00 g (2,3 mmol) Eicosapentaensäure-(2-tert.-butyloxycarbonyl- aminoethyl)-ester wurden in 40 ml Dichlormethan bei 20°C mit 2 ml (26,1 mmol) Trifluoressigsäure versetzt. Nach 2 h wurde die Reaktionsmischung mehrmals mit gesättigter NaHC0 -Lösung und
dreimal mit wenig Wasser gewaschen und über NaSO^ kurz getrock¬ net. Anschließend wurde bei 20°C 10 min gasförmiges HC1 einge¬ leitet und die Reaktionsmischung anschließend sofort unter redu¬ ziertem Druck bei 40°C eingeengt. Die Ausbeute betrug 0,83 g (97 %).1.00 g (2.3 mmol) of eicosapentaenoic acid (2-tert-butyloxycarbonylaminoethyl) ester was added to 40 ml of dichloromethane at 20 ° C with 2 ml (26.1 mmol) of trifluoroacetic acid. After 2 h, the reaction mixture was washed several times with saturated NaHC0 solution and washed three times with a little water and briefly dried over NaSO 4. Gaseous HC1 was then passed in at 20 ° C. for 10 min and the reaction mixture was then immediately concentrated at 40 ° C. under reduced pressure. The yield was 0.83 g (97%).
IH-NMR (300 MHz, CDC13), δ (ppm): 0.98(t, J=8Hz, 3H) ; 1.70(m,2H); 1.95-2.20(kB,4H); 2.45(t, =8Hz, 2H) ; 2.70-3.00(kB,8H) ; 3.35(s, br,2H): 4.42(s,br, 2H) ; 5.20-5.60(kB, 10H) ; 8.25(s,br, 3H) .IH NMR (300 MHz, CDC1 3 ), δ (ppm): 0.98 (t, J = 8Hz, 3H); 1.70 (m, 2H); 1.95-2.20 (kB, 4H); 2.45 (t, = 8Hz, 2H); 2.70-3.00 (kB, 8H); 3.35 (s, br, 2H): 4.42 (s, br, 2H); 5.20-5.60 (kB, 10H); 8.25 (s, br, 3H).
Beispiel 10Example 10
Docosahexaensäure-(2-dimeth laminoethyl)-ester-hydrochloridDocosahexaenoic acid (2-dimeth laminoethyl) ester hydrochloride
0,3 g (0,75 mmol) der Substanz des Beispiels 6 wurden in 20 ml Hexan nach AAV 3 mit gasförmiger HC1 umgesetzt. Die Ausbeute betrug 0,31 g (95 %) . 0.3 g (0.75 mmol) of the substance of Example 6 were reacted in 20 ml of hexane according to AAV 3 with gaseous HC1. The yield was 0.31 g (95%).
IH-NMR (300 MHZ, CDC13), δ (ppm): 0.98(t, J=8Hz,3H) ; 2.08(m,2H); 2.35-2.50(kB,4H) ; 2.70-3.10(kB, 16H) ; 3.35(t,J=6Hz, 2H) ; 4.80(t, J=6HZ,2H); 5.25-5.55(kB, 12H) ; 13.0(s,lH).IH-NMR (300 MHz, CDC1 3 ), δ (ppm): 0.98 (t, J = 8Hz, 3H); 2.08 (m. 2H); 2.35-2.50 (kB, 4H); 2.70-3.10 (kB, 16H); 3.35 (t, J = 6Hz, 2H); 4.80 (t, J = 6HZ, 2H); 5.25-5.55 (kB, 12H); 13.0 (s, lH).
Beispiel 11Example 11
Octadecatriensäure-(2-dimethylaminoethy1)-ester-hydrochloridOctadecatrienoic acid (2-dimethylaminoethyl) ester hydrochloride
0,7 g (2,0 mmol) der Substanz des Beispiels 7 wurden in 10 ml Hexan nach AAV 3 mit gasförmigem HC1 umgesetzt. Die Ausbeute betrug 0,77 g (100 %) .0.7 g (2.0 mmol) of the substance of Example 7 were reacted in 10 ml of hexane according to AAV 3 with gaseous HC1. The yield was 0.77 g (100%).
IH-NMR (250 MHZ, CDC13), δ (ppm): 1.00(t,J=8HZ, 3H) ; 1.15-1.40(kB, 8H); 1.55(m,2H); 1.90-2.10(kB,4H) ; 2.40(t, =8Hz, 2H) ; 2.65-2.85 (kB,4H); 2.90(d,J=6Hz,6H) ; 3.40(m,2H); 4.55(t, J=6Hz, 2H) ; 5.20-5.55(kB,6H); 12.10(s,lH).
In folgenden Versuchen wurde die Wirkung der neuen Verbindungen gezeigt:IH NMR (250 MHz, CDC1 3 ), δ (ppm): 1.00 (t, J = 8HZ, 3H); 1.15-1.40 (kB, 8H); 1.55 (m. 2H); 1.90-2.10 (kB, 4H); 2.40 (t, = 8Hz, 2H); 2.65-2.85 (kB, 4H); 2.90 (d, J = 6Hz, 6H); 3.40 (m. 2H); 4.55 (t, J = 6Hz, 2H); 5.20-5.55 (kB, 6H); 12.10 (s, lH). The effects of the new compounds were shown in the following experiments:
Versuch 1: Einfluß auf die Granulozyten-AktivierungExperiment 1: Influence on granulocyte activation
Granulozyten stellen den dominierenden Zelltyp unter den Leuko¬ zyten-Populationen des Menschen dar und sind insbesondere am frühen als auch am späten Organversagen (early/late organ failure) bei Sepsis und Trauma beteiligt. Es sind die Zellen, die sich aktiv als erste in einem Entzündungsherd anreichern und die erste Reihe der antimi robiellen Abwehr bilden. Die hohe Reakti¬ vität dieses Zelltyps auf alle möglichen inflammatorisehen Stimuli trägt stark zur hohen Lethalität in ARDS (=Adult Respi- ratory Distress Syndrome; Schocklunge) und beim septischen Schock bei [Infect. Immun. 50, 527 (1985); Prostaglandins 27, 227Granulocytes represent the dominant cell type among the leukocyte populations of humans and are particularly involved in early and late organ failure (early / late organ failure) in sepsis and trauma. It is the cells that are the first to accumulate actively in an inflammatory focus and form the first row of the antimicrobial defense. The high reactivity of this cell type to all possible inflammatory stimuli contributes strongly to the high lethality in ARDS (= Adult Respiratory Distress Syndrome; shock lung) and in septic shock [Infect. Immune. 50, 527 (1985); Prostaglandins 27, 227
(1987)]. Stimulierte Granulozyten sind charakterisiert durch die Freisetzung einer großen Zahl lytischer Enzyme und insbesondere von großen Mengen an verschiedenen Sauerstoffradikalen.(1987)]. Stimulated granulocytes are characterized by the release of a large number of lytic enzymes and, in particular, large amounts of various oxygen radicals.
Daher wurde zur Beurteilung der omega-3-Fettsäurederivate zu¬ nächst die He mwirkung auf mit verschiedenen Stimuli aktivierte neutrophile Granulozyten in einem Chemilumineszenz Assay ge¬ messen. Die Chemilumineszenz Technik ist ein sehr sensitives Detektionssystem für Sauerstoffradikale.Therefore, in order to assess the omega-3 fatty acid derivatives, the inhibitory effect on neutrophil granulocytes activated with various stimuli was first measured in a chemiluminescence assay. The chemiluminescence technique is a very sensitive detection system for oxygen radicals.
Zur Isolierung von PMN aus menschlichem Vollblut [J. Clin. Invest. 77, 925 (1986)] und Messung der Aktivität wurden poly- morphonukleäre Neutrophile aus heparinisiertem Vollblut gesunder Spender durch Dextran Sedimentation (Dextran T250, 3 %; 1 x g; 1 h bei Raumtemperatur) und anschließender Auftrennung über einem Percoll-Zweistufen-Gradienten (60 %/68 % PERCOLL in HBSS ohne Caz+/Mgz+) mit 600 x g über 30 min bei 4°C, gewonnen. Die Zell¬ fraktion aus der 60 %/68 % PERCOLL Zwischenphase wurden zweimal gewaschen und bestand zu > 98 % aus neutrophilen Granulozyten. Die Che ilumineszenz-Messungen [Meth. Enzym. 133, 449 (1986)] wurden in einem BIOLUMAT 9505 (Fa. Berthold, Wildbad) bei 37°C durchgeführt. Die Proben setzten sich wie folgt zusammen: Lucigenien 10 μM, HBSS mit Ca2+/Mg2+ je 1 mM und 0,2 % HSA in einem Gesamtvolumen von 0,5 ml. Stimuli (wie TNF, PAF, fMLP oder
PMA) wie auch Inhibitoren (mehrfach ungesättigte Fettsäurederi¬ vate) in verschiedenen Konzentrationen wurden in 20 μl Volumina zugesetzt. Die Ergebnisse wurden als kumulierte Chemilumineszenz- Impulse über eine Periode von 30 min gewonnen, die prozentuale Hemmung bezogen auf den verwendeten Stimulus errechnet und daraus die halbmaximale Hemmkonzentration bestimmt.For the isolation of PMN from human whole blood [J. Clin. Invest. 77, 925 (1986)] and measurement of the activity, polymorphonuclear neutrophils from heparinized whole blood from healthy donors were subjected to dextran sedimentation (Dextran T250, 3%; 1 xg; 1 h at room temperature) and subsequent separation using a Percoll two-stage gradient ( 60% / 68% PERCOLL in HBSS without Ca z + / Mg z + ) at 600 xg over 30 min at 4 ° C. The cell fraction from the 60% / 68% PERCOLL intermediate phase was washed twice and consisted of> 98% neutrophil granulocytes. The chiluminescence measurements [Meth. Enzyme. 133, 449 (1986)] were carried out in a BIOLUMAT 9505 (from Berthold, Wildbad) at 37 ° C. The samples were composed as follows: Lucigenien 10 μM, HBSS with Ca 2+ / Mg 2+ each 1 mM and 0.2% HSA in a total volume of 0.5 ml. Stimuli (such as TNF, PAF, fMLP or PMA) as well as inhibitors (polyunsaturated fatty acid derivatives) in various concentrations were added in 20 μl volumes. The results were obtained as cumulative chemiluminescence pulses over a period of 30 min, the percentage inhibition based on the stimulus used and the half-maximal inhibitory concentration determined therefrom.
Tabelle 1 zeigt, daß die neuen Substanzen bei 5 min. Vorinkuba¬ tion eine gute Hemmwirkung auf die Aktivierung von humanen poly- morphonukleären Neutrophilen besitzen. Die Beispiele 1 und 8 zeigen, daß die Esterform eine deutlich bessere Hemmwirkung aus¬ übt als die Esterkomponenten alleine bzw. als eine äquimolare Mischung der Säure- und der Aminoalkoholkomponente. Die Referenzsubstanz Pentoxifyllin ergibt ebenfalls geringere Hemmeffekte.Table 1 shows that the new substances at 5 min. Pre-incubation has a good inhibitory effect on the activation of human polymorphonuclear neutrophils. Examples 1 and 8 show that the ester form has a significantly better inhibitory effect than the ester components alone or as an equimolar mixture of the acid and the amino alcohol component. The reference substance pentoxifylline also results in lower inhibitory effects.
Tabelle 1Table 1
Hemmung der Aktivierung von humanen polymorphonukleären Neutro- philen (PMN) nach Stimulierung der Zellen mit rHuTNF (1 ng/ml)Inhibition of the activation of human polymorphonuclear neutrophils (PMN) after stimulation of the cells with rHuTNF (1 ng / ml)
Substanz des Beispiels IC50 [μM]Substance of the example IC50 [μM]
1 16.51 16.5
2 11.0 4 7.52 11.0 4 7.5
6 17.46 17.4
7 6.27 6.2
8 6.78 6.7
9 24.3 10 8.19 24.3 10 8.1
11 11.211 11.2
Eicosapentaensäure (EPA) > 100Eicosapentaenoic acid (EPA)> 100
Dimethylaminoethanol (DMAE) 44.5Dimethylaminoethanol (DMAE) 44.5
EPA + DMAE (äquimolar) 39.0 Pentoxifyllin 72.0EPA + DMAE (equimolar) 39.0 pentoxifylline 72.0
Tabelle 2 zeigt, daß die Substanz des Beispiels 8 eine hohe Wirk¬ stärke gegen andere natürliche Stimuli (PAF/fMLP) der PMN-Akti- vierung besitzt, nicht jedoch gegen den Phorbolester und PKC-Aktivator PMA.
Tabelle 2Table 2 shows that the substance of Example 8 has a high potency against other natural stimuli (PAF / fMLP) of PMN activation, but not against the phorbol ester and PKC activator PMA. Table 2
Hemmung der Stimulierung von polymorphonukleären Neutrophilen (PMN)Inhibition of stimulation of polymorphonuclear neutrophils (PMN)
Vergleich der Wirkung bei verschiedenen StimuliComparison of the effects with different stimuli
Stimulus Inhibitor IC50 [μM] rHuTNF Substanz des Beispiels 8 6.7 fMLP Substanz des Beispiels 8 10.0Stimulus inhibitor IC50 [μM] rHuTNF substance of Example 8 6.7 fMLP substance of Example 8 10.0
PAF Substanz des Beispiels 8 14.5PAF substance of Example 8 14.5
PMA Substanz des Beispiels 8 > 50PMA substance of Example 8> 50
Tabelle 3 zeigt die Überlegenheit des Beispiels 8 gegenüber dem Tri-EPA-Glyzerin in Bezug auf schnelle Entfaltung der Hemmwirkung in humanen PMN. Der schnelle Effekt auf die Radikalfreisetzung ist weder auf etwaige Radikalfängereigenschaften (Kontrolle im zellfreien System negativ) noch auf eine zytotoxische Wirkung der Testsubstanz auf die Effektorzellen zurückzuführen. Die Viabili- tat nach 4 h Vorinkubation und 30minütiger Exposition gegen TNF war stets > 90 %.
Table 3 shows the superiority of Example 8 over the tri-EPA glycerol in terms of rapid development of the inhibitory effect in human PMN. The rapid effect on the radical release is neither due to any radical scavenger properties (control in the cell-free system negative) nor to a cytotoxic effect of the test substance on the effector cells. The viability after 4 h of pre-incubation and 30 min exposure to TNF was always> 90%.
Tabel le 3Table 3
Hemmung der Granulozyten - StimulierungInhibition of granulocyte stimulation
Abhängigkeit von der VorinkubationsdauerDependence on the pre-incubation period
Vorinkubation Konzentration Hemmung (in %, bezogen aufPre-incubation concentration inhibition (in%, based on
TNF-behandelte Zellkontrolle)TNF-treated cell control)
T [h] [μM] Beispiel 8 Tri-EPA-Glyzerin 0 20T [h] [μM] Example 8 Tri-EPA glycerol 0 20
55
0.5 200.5 20
55
2020th
55
2 20 52 20 5
3 203 20
55
4 204 20
* Es wurde keine Hemmung sondern eine Stimulierung beobachtet* No inhibition but stimulation was observed
Versuch 2: Hemmung der Lymphozyten ProliferationExperiment 2: Inhibition of Lymphocyte Proliferation
Zur Evaluierung der Wirkung der omega-3-Fettsäuren und ihrer Derivate auf die Lymphoproliferation wurden Splenozyten von Balb/c Mäusen verwendet. Stimulierung der Splenozytenkultur mit LPS führt zur Proliferation der B-Zell Subpopulation, Stimulie¬ rung mit Enterotoxin B zur T-Zellproliferation.
Die Splenozytenkulturen wurden 2 h mit verschiedenen Konzentra¬ tionen der Testsubstanzen in Mi rotiterplatten vorinkubiert und anschließend mit LPS bzw. Enterotoxin B zur Proliferation indu¬ ziert. Die relative Wachstumshemmung, bezogen auf die entspre¬ chende Kontrolle ohne Inhibitor, wurde aus der Zählausbeute des eingebauten [3H]-Thymidin nach zwei Tagen Inkubation bei 37°C und anschließendem 6 stündigem 3H-Tymidinpulse (1 μCi/ 200 μl/well) berechnet und danach die halbmaximale Hemmkonzentrationen (IC50) bestimmt.Splenocytes from Balb / c mice were used to evaluate the effect of omega-3 fatty acids and their derivatives on lymphoproliferation. Stimulation of the splenocyte culture with LPS leads to the proliferation of the B cell subpopulation, stimulation with Enterotoxin B to the T cell proliferation. The splenocyte cultures were preincubated with different concentrations of the test substances in microtiter plates for 2 hours and then induced with LPS or enterotoxin B for proliferation. The relative growth inhibition, based on the corresponding control without inhibitor, was calculated from the count yield of the incorporated [3H] -thymidine after two days of incubation at 37 ° C. and then a 6-hour 3H-tymidine pulse (1 μCi / 200 μl / well) and then determined the half-maximum inhibitory concentrations (IC50).
Tabelle 4 zeigt für Eicosapentaensäure und für die Ester der Beispiele 1 und 8 einen gleich stark ausgeprägten antiprolife- rativen Effekt sowohl auf die B- wie T-Zellsubpopulation von Maus-SplenozytenTable 4 shows for eicosapentaenoic acid and for the esters of Examples 1 and 8 an equally pronounced antiproliferative effect on both the B and T cell subpopulation of mouse splenocytes
Tabelle 4Table 4
Splenozyteπ ProliferationSplenocyte proliferation
Effekt von EPA und EPA-DerivatenEffect of EPA and EPA derivatives
Versuch 3: Effekt der Testsubstanzen im EndotoxinschockExperiment 3: Effect of the test substances in endotoxin shock
Die Sensitivitat von Mäusen gegen Lipopolysaccharid ist abhängig von Stamm und Alter der Tiere. Vorversuche mit Balb/c Mäusen ergaben eine lethale Dosis von 20 mg/kg i.v. bei 6-8 Wochen alten Tieren.The sensitivity of mice to lipopolysaccharide depends on the strain and age of the animals. Preliminary experiments with Balb / c mice gave a lethal dose of 20 mg / kg IV. in 6-8 week old animals.
Der Effekt der Testsubstanzen im Endotoxinschock wurde unter¬ sucht, indem die Inhibitoren 30 min vor der LPS-Gabe (20 mg/Kg) i.v. appliziert wurden.The effect of the test substances in endotoxin shock was investigated by i.v. the inhibitors 30 min before LPS administration (20 mg / kg). were applied.
In diesen Versuchen zeigten die neuen Verbindungen und insbe¬ sondere die Substanz des Beispiels 8 sehr gute Wirkungen.
Tabelle 5 zeigt die protektiven Effekte der Substanzen im Ver¬ gleich zu Eicosapentaensäure und Eicosapentaensäuretriglycerid. Die Tiere (Balb/c Mäuse, 6-8 Wochen alt) wurden mit verschiedenen Dosen der Beispielsubstanzen einmal (Bolus, i.v.) behandelt. 30 min später erfolgte eine i.v. Applikation mit 20 mg/kg Lipo¬ polysaccharid (LPS) aus E. coli, Serotyp 0 111 B4. Diese Dosis erwies sich in der Kontrollgruppe als 100 % lethal. Pro Dosis der Testsubstanzen wurden je 5-10 Tiere in mindestens 2 unabhängigen Experimenten getestet. In der Tabelle ist die prozentuale über- lebensrate für den angegebenen Dosisbereich aufgelistet.In these experiments the new compounds and in particular the substance of Example 8 showed very good effects. Table 5 shows the protective effects of the substances in comparison with eicosapentaenoic acid and eicosapentaenoic acid triglyceride. The animals (Balb / c mice, 6-8 weeks old) were treated once with different doses of the sample substances (bolus, iv). 30 min later, an iv application with 20 mg / kg lipopolysaccharide (LPS) from E. coli, serotype 0 111 B4 was carried out. This dose was found to be 100% lethal in the control group. For each dose of the test substances, 5-10 animals were tested in at least 2 independent experiments. The table shows the percentage survival rate for the specified dose range.
Tabelle 5Table 5
Protektion gegen lethale LPS-Effekte in vivoProtection against lethal LPS effects in vivo
Maximale Protektion nach 48 h überlebende Balb/c Dosis zur Pro- [%] Mäuse tektion [mg/kg]Maximum protection after 48 h surviving Balb / c dose for pro [%] mouse detection [mg / kg]
Substanz des Beispiels 1 80-100 10-20Substance of Example 1 80-100 10-20
Substanz des Beispiels 2 60- 80 20Substance of Example 2 60-80 20
Substanz des Beispiels 4 80 20Substance of Example 4 80 20
Substanz des Beispiels 5 60 10-20Substance of Example 5 60 10-20
Substanz des Beispiels 7 80-100 20 Substanz des Beispiels 8 80-100 5-20Substance of Example 7 80-100 20 Substance of Example 8 80-100 5-20
Substanz des Beispiels 9 60 20Substance of Example 9 60 20
Eicosapentaensäure (EPA) 40- 60 20Eicosapentaenoic acid (EPA) 40-60 20
Tri-EPA-Glycerin 60- 80 10-20Tri-EPA glycerin 60-80 10-20
Die in der Beschreibung verwendeten Abkürzungen haben folgende Bedeutungen:The abbreviations used in the description have the following meanings:
DC DünnschichtchromatographieTLC thin layer chromatography
EtOH Ethanol fMLP Formyl-Methionyl-Leucyl-PhenylalaninEtOH ethanol fMLP formyl-methionyl-leucyl-phenylalanine
HBSS Hanks balanced salt solutionHBSS Hanks balanced salt solution
Hex n-HexanHex n-hexane
HSA Human-Serumalbumin i.v. intravenös MTB Methyl-tert.-butyl-ether
NMR Kernresonanzspektroskopie s=Singulett, d=Dublett, t=Triplett, q=Quartett, m=Multiplett, kB=komplexer Bereich, br=breitHSA human serum albumin intravenously MTB methyl tert-butyl ether NMR nuclear magnetic resonance spectroscopy s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, kB = complex range, br = broad
LPS LipopolysaccharidLPS lipopolysaccharide
PAF Platelet Activating FactorPAF Platelet Activating Factor
PMA Phorbol-12-myristat-13-acetatPMA phorbol-12-myristate-13-acetate
PMN Polymorphonucleare neutrophile LeukozytenPMN polymorphonuclear neutrophil leukocytes
TNF Tumornekrosefaktor
TNF tumor necrosis factor
Claims
1. Verbindungen der Formel I1. Compounds of formula I.
Rl-X-Y- NR2R3 I,Rl-X-Y- NR2R3 I,
worinwherein
Rl der Acylrest von 5,8, 11, 14, 17-Eicosapentaensäure, 4, 7, 10, 13, 16, 19-Docosahexaensäure oder 9, 12, 15-Octa- decatriensäure,R1 is the acyl radical of 5,8, 11, 14, 17-eicosapentaenoic acid, 4, 7, 10, 13, 16, 19-docosahexaenoic acid or 9, 12, 15-octa-decatrienoic acid,
X ein Sauerstoffatom oder ein NH-Gruppe,X is an oxygen atom or an NH group,
Y eine C2_3-Alkylengruppe,Y is a C 2 _ 3 alkylene group,
R2 und R3 Wasserstoffatome oder Cι_2-AlkylgruppenR2 and R3 are hydrogen atoms or Cι_2 alkyl groups
darstellen, sowie deren Salze mit physiologisch verträg- liehen Säuren und Cι_2-Trialkylammoniumsalze.represent, as well as their salts with physiologically tolerated acids and Cι_2-trialkylammonium salts.
2. Verbindungen der Formel I gemäß Anspruch zur Verwendung bei der Bekämpfung von Krankheiten. 2. Compounds of formula I according to claim for use in combating diseases.
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DE19914120916 DE4120916A1 (en) | 1991-06-25 | 1991-06-25 | OMEGA-3 MULTIPLE UNSATURATED FAT SEA DERIVATIVES, THEIR PREPARATION AND USE |
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Family
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CN104119242A (en) * | 2008-10-09 | 2014-10-29 | 泰米拉制药公司 | Improved amino lipids and methods for the delivery of nucleic acids |
CN104119242B (en) * | 2008-10-09 | 2017-07-07 | 泰米拉制药公司 | The amino lipids of improvement and the method for delivering nucleic acid |
US9896316B2 (en) | 2016-06-30 | 2018-02-20 | The Procter & Gamble Company | End effector for a transport device for the movement of parent rolls of convolutely wound web materials |
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DE4120916A1 (en) | 1993-01-07 |
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