WO1993000326A1

WO1993000326A1 - Omega-3 polyunsaturated fatty-acid derivatives, their preparation and their use

Info

Publication number: WO1993000326A1
Application number: PCT/EP1992/001337
Authority: WO
Inventors: Bernhard Bockstiegel; Franz Emling; Barbara Jessel; Peter Lechtken; Ulrich Hoercher
Original assignee: Basf Aktiengesellschaft
Priority date: 1991-06-25
Filing date: 1992-06-13
Publication date: 1993-01-07
Also published as: MX9203292A; DE4120916A1

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 polyunsaturated fatty acid derivatives, their production and use

description

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.

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)].

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)].

The 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, p. 47].

Compounds with better properties have now been found.

The invention relates to compounds of the formula I.

Rl-X-Y- NR2R3 I,

wherein

Ri is the acyl residue of 5,8, 11, 14, 17-eicosapentaenoic acid,

4,7,10, 13,16, 19-docosahexaenoic acid or 9, 12, 15-0ctadecatrienoic acid,

X is an oxygen atom or an NH group,

Y is a _C2-C3 alkylene group,

R2 and R3 are hydrogen atoms or Cι_2 alkyl groups

represent, and their salts with physiologically acceptable acids and Cι_2-trialkylammonium salts.

5, 8, 11, 14, 17-egg cosapentaenoic acid (= EPA) has the formula

-COOH,

4, 7, 10, 13, 16, 19-docosahexaenoic acid (= DHA) the formula OOH and

9, 12, 15-octadecatrienoic acid (= o-linolenic acid) the formula COOH.

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.

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,

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.

The 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 exhaustively alkylated ammonium compounds are at

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.

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.

Depending on the indication, 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. 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:

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.

The examples listed below describe the invention in detail, but in no way limit the invention.

Manufacturing regulations:

General labor regulations (AAV):

AAV 1: Production of the carboxylic acid chlorides:

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: Preparation of the carboxylic acid esters and carboxamides:

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:

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.

Special working regulations

example 1

Eicosapentaenoic acid (2-dimethylaminoethyl) ester

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, 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).

Example 2

Eicosapentaenoic acid (2-dieth laminoethy1) ester

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, 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).

Example 3

Eicosapentaenoic acid (3-dimethylaminopropyl) ester

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).

Example 4

Eicosapentaenoic acid (l-methyl-2-dimethylaminoethyl) ester

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).

Example 5

Egg cosapentaenoic acid (2-d imethy 1 ami noethy 1) -ami d

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, 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).

Example 6

Docosahexaenoic acid (2-dimethylaminoeth l) ester

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).

Example 7

Octadecatr ienoic acid (2-d imethy 1 ami noethy 1) ester

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).

Example 8

Eicosapentaenoic acid (2-dimeth 1aminoethyl) ester hydrochloride

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, 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

Eicosapentaenoic acid (2-aminoeth 1) ester hydrochloride

1. N-tert-butyloxycarbonyl-2-aminoethanol

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. Eicosapentaenoic acid (2-tert-butyloxycarbonylaminoethyl) ester

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, 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. Eicosapentaenoic acid (2-aminoethyl) ester hydrochloride

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, 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).

Example 10

Docosahexaenoic acid (2-dimeth laminoethyl) ester hydrochloride

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, 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).

Example 11

Octadecatrienoic acid (2-dimethylaminoethyl) ester hydrochloride

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, 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:

Experiment 1: Influence on granulocyte activation

Granulocytes 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)]. Stimulated granulocytes are characterized by the release of a large number of lytic enzymes and, in particular, large amounts of various oxygen radicals.

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.

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.

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.

Table 1

Inhibition of the activation of human polymorphonuclear neutrophils (PMN) after stimulation of the cells with rHuTNF (1 ng / ml)

Substance of the example IC50 [μM]

1 16.5

2 11.0 4 7.5

6 17.4

7 6.2

8 6.7

9 24.3 10 8.1

11 11.2

Eicosapentaenoic acid (EPA)> 100

Dimethylaminoethanol (DMAE) 44.5

EPA + DMAE (equimolar) 39.0 pentoxifylline 72.0

Table 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

Inhibition of stimulation of polymorphonuclear neutrophils (PMN)

Comparison of the effects with different stimuli

Stimulus inhibitor IC50 [μM] rHuTNF substance of Example 8 6.7 fMLP substance of Example 8 10.0

PAF substance of Example 8 14.5

PMA substance of Example 8> 50

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%.

Table 3

Inhibition of granulocyte stimulation

Dependence on the pre-incubation period

Pre-incubation concentration inhibition (in%, based on

TNF-treated cell control)

T [h] [μM] Example 8 Tri-EPA glycerol 0 20

5

0.5 20

5

20th

5

2 20 5

3 20

5

4 20

5

* No inhibition but stimulation was observed

Experiment 2: Inhibition of Lymphocyte Proliferation

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).

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

Table 4

Splenocyte proliferation

Effect of EPA and EPA derivatives

Eicosapentaenoic acid Example 1 Example 8

Experiment 3: Effect of the test substances in endotoxin shock

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.

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 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.

Table 5

Protection against lethal LPS effects in vivo

Maximum protection after 48 h surviving Balb / c dose for pro [%] mouse detection [mg / kg]

Substance of Example 1 80-100 10-20

Substance of Example 2 60-80 20

Substance of Example 4 80 20

Substance of Example 5 60 10-20

Substance of Example 7 80-100 20 Substance of Example 8 80-100 5-20

Substance of Example 9 60 20

Eicosapentaenoic acid (EPA) 40-60 20

Tri-EPA glycerin 60-80 10-20

The abbreviations used in the description have the following meanings:

TLC thin layer chromatography

EtOH ethanol fMLP formyl-methionyl-leucyl-phenylalanine

HBSS Hanks balanced salt solution

Hex n-hexane

HSA 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 lipopolysaccharide

PAF Platelet Activating Factor

PMA phorbol-12-myristate-13-acetate

PMN polymorphonuclear neutrophil leukocytes

TNF tumor necrosis factor

Claims

1. Compounds of formula I.

Rl-X-Y- NR2R3 I,

wherein

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 is an oxygen atom or an NH group,

Y is a C ₂ _ ₃ alkylene group,

R2 and R3 are hydrogen atoms or Cι_2 alkyl groups

represent, as well as their salts with physiologically tolerated acids and Cι_2-trialkylammonium salts.

2. Compounds of formula I according to claim for use in combating diseases.