RU2378284C2 - Method of producing n-acyl derivatives of amino acids (versions) - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: present invention relates to novel methods (versions) of producing N-acyl derivatives of amino acids of general formula

, where n equals 2 or 3; and R is

or their salts, using anhydrides of glutaric or succinic acid.

EFFECT: advantages of the proposed methods lie in simplicity of their realisation, easy extraction of the end product and high output of the end products.

2 cl, 27 tbl, 22 ex

Description

The present invention relates to the field of bioorganic chemistry and relates to new methods for the synthesis of N-acyl derivatives of amino acids and their pharmaceutically acceptable salts, used as anti-allergic, anti-inflammatory and lipid-lowering agents.

State of the art

As you know, at present allergic diseases and lipid metabolism disorders are very common due to poor environmental conditions, changes in the nutritional structure and lifestyle of the population. Therefore, the problem of creating drugs to combat these pathologies, as well as with inflammatory processes, usually accompanying allergies, continues to be relevant.

In this regard, compounds including residues of substances of natural origin are of particular interest, since lower toxicity and incidence of side effects can be predicted for them.

The publication of international application WO 99/01103 describes the antiallergic and hypolipidemic effect of N-acyl derivatives of biogenic amines, for example, γ-glutamylhistamine, and its closest analogue, glutaryl histamine, which are closest in structure and effect to the claimed compounds. In the article Krzhechkovskaya V.V., Zheltukhin G.A., Nebolsin V.E. et al. Study of antianaphylactic activity and mechanisms of action of γ-L-glutamylhistamine. // Pathogenesis, 2003, T. 1, No. 2, p. 60-64, it was shown that γ-glutamylhistamine has a pronounced antianaphylactic activity when using various animal species and methods of administration. The results obtained indicate that in the mast cells of animals under the influence of γ-glutamylhistamine significantly decreases the content of histamine and its antigen-stimulated secretion. In a test to study the effect of glutaryl histamine on the severity of antigen-induced bronchospasm, a decrease in bronchospasm of more than 50% was shown compared with the control. This effect was manifested both in the oral and intratracheal methods of its administration in a low dose of 50 μg / kg. Glutaryl histamine has the ability to reduce the manifestations of passive cutaneous anaphylaxis from 34 to 42% and, thus, is superior in effectiveness to suprastin, but inferior to clarithin. WO 99/01103 shows that when animals were administered glutaryl histamine at doses of 50 and 500 μg / kg, a significant decrease in delayed-type hypersensitivity was demonstrated. In addition, glutaryl histamine in doses of 50 and 500 μg / kg also had some anticholesterol-mimic activity, reducing total cholesterol compared to animals with atherogenic load by 5-7%.

The disadvantage of glutaryl histamine is its relatively high cost and low availability of the starting material for its production - histamine. In addition, the specified substance is not effective enough in the above tests.

In order to expand the arsenal of technical means and create a more effective and affordable anti-allergic, anti-inflammatory and lipid-lowering drugs, the inventors have identified some specific N-acyl derivatives of amino acids of the general formula (I) disclosed in the publication of international application WO 99/01103, but specifically in it not described, not received and not characterized.

Thus, the compounds of the present invention N ^α -succinyl-L-tryptophan (II), N ^α -glutaryl-L-tryptophan (III), N ^α -glutaryl-L-histidine (IV) fall under the general formula (I) of the above international application and N ^α -succinyl-L-histidine (V). However, in this publication neither specific structural formulas of these compounds nor any physicochemical characteristics are given, nor are methods for their preparation described. International Publication WO 03/072124 discloses formulas of N ^α -glutaryl-L-tryptophan (II), N ^α -glutaryl-L-histidine (IV) and N ^α -succinyl-L-histidine (V), but the method is not described their synthesis and not given their physico-chemical constants.

One of the compounds glutaryl histidine is mentioned only in the form of methyl ester at the C-terminus of His [Glt-His (OMe)] in US patent 3963691, as an intermediate in the synthesis of the Glt-His-Trp-Ser-Tyr-Gly-Leu-Arg peptide -Pro-Gly-poly-Lys.

The compound succinylhistidine is mentioned in the publication of international application WO 93/04690. This publication indicates that the addition of free imidazole or succinylhistidine accelerates the interaction of carnosine with dihydroxyacetone. Methods for the synthesis of succinylhistidine and its physicochemical constants are not given.

The structural formula of succinyltryptophan is mentioned in US application 2005079515 . In this publication, neither the method of synthesis of succinyltryptophan, nor its physicochemical constants are given.

Preparation of N ^α glutaryl-L-histamine is described in International Patent Publication WO 99/01103 and is a N-acylation of biogenic amine with glutaric anhydride in anhydrous N, N-dimethylformamide. In addition, in the publication Gershkovich A.A., Kiberev V.K. // Chemical synthesis of peptides. / Kiev: Naukova Dumka, 1992, 360, a method for the acylation of amino acids in an aqueous-organic, highly alkaline medium is described.

In Sorm F., Pravda Z., Proteins and amino acids. X. Synthesis of two peptide analogs. // Chemicke Listy pro Vedu a Prumysl., 1951, V.45, p.423-425, describes a method for synthesizing succinyl tyrosine ethyl ether in a mixture of water and ethyl acetate at a ratio of (1: 1) based on tyrosine ethyl chloride and glutaric anhydride in the presence of NaHCO ₃ to maintain a slightly alkaline pH.

However, acylation of free histidine anhydrides of dicarboxylic acids is not described in the literature.

The aim of the present invention are new methods for the synthesis of effective N-acyl derivatives of amino acids with anti-allergic, anti-inflammatory and lipid-lowering effects in low doses and without side effects.

The inventors have developed a simple and preparative method for the synthesis of compounds of general formula (I), which consists in the fact that the anhydride of glutaric or succinic acid in the form of a solid is added to an aqueous solution of an amino acid in the absence of an inorganic and organic base to obtain the target product with a rather high yield of 55- 60%

The inventors have also developed another method for the synthesis of compounds of general formula (I), including N-acyl derivatives of histidine, comprising carrying out the reaction in a two-phase system consisting of an aqueous solution of histidine and a solution of anhydride in a suitable organic solvent using an excess of acylating agent.

SUMMARY OF THE INVENTION

The present invention also relates to a method for producing N-acyl derivatives of amino acids of the general formula I

where n is 2 or 3; and

R represents

and their salts, including the addition of glutaric or succinic anhydride as a solid to an aqueous solution of an amino acid of the general formula:

or its salt, where

R represents

and optionally, converting the desired product into its salt.

The present invention also relates to a method for producing N-acyl derivatives of amino acids of the general formula I

where n is 2 or 3; and

R represents

and their salts, including carrying out the reaction in a two-phase system with glutaric or succinic acid anhydride in a water-immiscible organic solvent with an aqueous solution of an amino acid of the general formula:

or its salt, where

R represents

and, optionally, converting the desired product into its salt.

DETAILED DESCRIPTION OF THE INVENTION

Preferred compounds of the general formula I are presented in table 1.

Table 1 Compound Connection No. R n N ^α- succinyl-L-tryptophan II

(Ind)

(Ind) 2 N ^α -glutaryl-L-tryptophan III Ind 3 N ^α -glutaryl-L-histidine IV

(Im) 3 N ^α- succinyl-L-histidine V Im 2

The synthesis of compounds of general formula I can be carried out in two ways. The first method consists in gradually adding to the aqueous solution the amino acids of the general formula

or its salt, where

R represents

glutaric or succinic anhydride in the form of a solid, followed by isolation of the target product by ion exchange chromatography, preferably by passing the reaction mixture through a column of cation exchange resin and subsequent crystallization from an aqueous solution. The resulting crystals of the expected product are washed with a suitable solvent, preferably methanol. The main advantage of the proposed method is the absence of alkali in an aqueous solution of an amino acid, which prevents the inactivation of dicarboxylic acid anhydride as a result of hydrolysis. In addition, the imidazole residue in the amino acid molecule can carry out acid-base autocatalysis of the amino acid amino acid acylation reaction. Quite high yields (55-60%) when using the proposed method are achieved, in particular, due to the gradual addition of dicarboxylic acid anhydride, taken in excess, and intensive mixing of the reaction mass.

Compounds of general formula I can also be prepared by an alternative method in a two-phase system, comprising adding glutaric or succinic anhydride in a water-immiscible organic solvent to an aqueous solution of an amino acid of the general formula:

or its salt, where

R represents

This method allows you to use the excess acylating agent, to achieve complete acylation of the α-amino group of the amino acid and the yield of the target product of about 70%. To maintain the required pH, an organic base is used instead of inorganic alkali - pyridine, which does not hydrolyze the anhydride, and, moreover, is known to be an acylation catalyst. The use of pyridine avoids contamination of the final product with inorganic salts, which, together with the reaction product, remain in the aqueous layer. The approaches used make it possible to simplify the separation of the target product from unreacted anhydride and the corresponding amino acid and isolate the target product by simple crystallization.

Preferred water-immiscible organic solvents are butanol, ethyl acetate, chloroform.

Preferred solvents used to crystallize the target product are water-alcohol mixtures, in particular water-ethanol.

Compounds of general formula I can also be prepared as pharmaceutically acceptable salts with sodium hydroxide, potassium hydroxide, magnesium carbonate, lithium hydroxide, calcium carbonate by routine methods widely described in the literature.

The compounds of general formula I have anti-allergic, anti-inflammatory and lipid-lowering activity and can be used to treat allergic, anaphylactic, including diseases accompanied by inflammation, as well as impaired lipid metabolism.

In particular, the compounds of the present invention can be used to treat the following allergic diseases: bronchial asthma, allergic rhinitis, pollinosis, seasonal rhinitis, perennial rhinitis, atopic dermatitis, psoriasis, urticaria, allergic (including anaphylactic) reactions to insect bites and drugs , cold allergies, allergic conjunctivitis, chronic obstructive pulmonary diseases, namely chronic obstructive bronchitis, emphysema, bronchitis obliterans one of cystic fibrosis, as well as diseases related to lipid metabolism disorders, such as atherosclerosis, obesity, coronary heart disease and cerebral disease, myocardial infarction, stroke.

The compounds of the present invention are administered in an effective amount that provides the desired therapeutic result.

For the treatment of allergic diseases, including bronchial asthma, allergic rhinitis, hay fever, seasonal rhinitis, perennial rhinitis, atopic dermatitis, psoriasis, urticaria, allergic (including anaphylactic) reactions to insect bites and drugs, cold allergies, allergic conjunctivitis, chronic obstruction lung diseases, namely chronic obstructive bronchitis, emphysema, obliterating bronchitis, cystic fibrosis, as well as diseases associated with impaired lipid metabolism, such as atherosclerosis, obesity, ischemic heart and cerebral disease, myocardial infarction, stroke, compounds of general formula I may be administered orally, topically, parenterally, intranasally, by inhalation and rectally in dosage unit formulations containing non-toxic pharmaceutically acceptable carriers. As used herein, the term “parenteral administration” means subcutaneous, intravenous, intramuscular or intrathoracic injection or infusion.

The compounds of the present invention can be administered to a patient in doses of 0.01 to 10 mg / kg body weight per day, preferably in doses of 0.05 to 5 mg / kg one or more times per day.

It should be noted that the specific dose for each particular patient will depend on many factors, including the activity of the compound used, age, body weight, gender, general health status and diet of the patient, time and method of administering the drug, speed of its removal from an organism, the combination of drugs used specifically, and the severity of the disease in the individual being treated.

A detailed description of the compounds of the present invention, their preparation and study of pharmacological activity is presented in the following examples, intended to illustrate the preferred variants of the invention and not limiting its scope.

Examples of the synthesis of N-acyl derivatives of amino acids of the general formula (I)

The individuality of the obtained compounds was checked by TLC on Kieselgel 60 F ₂₅₄ Merck plates (Germany) in systems: methanol (1), chloroform-methanol-ammonia (4: 3: 1) (2).

Chromatograms were shown with chlorotolidine reagent, ninhydrin, iodine by luminescence in UV light.

The angles of optical rotation were measured on a Perkin Elmer 341 polarimeter (Sweden).

¹ H-NMR was recorded on an AMX-400 Bruker instrument (Germany).

The melting point was determined on a Boetius instrument (Germany).

Analytical HPLC was performed on a System Gold instrument (Beckman, USA): elution rate 0.25 ml / min, detection at 214 nm under conditions: Ultrasphere ODS Beckman column, 2 × 250 mm, 5 μm, elution 0 , 1% TFA, elution rate 0.25 ml / min (1); elution rate 1 ml / min, detection at 220 nm, Luna-5 Phenomenex column, C ₁₈ , 250 × 4.6 mm, elution with 25% acetonitrile in 0.05 M phosphate buffer (pH 3.0) (2) .

Example 1

N ^α -Glutaryl-L-histidine (IV)

Method A

To a solution of 103.4 g (0.67 mol) of histidine in 400 ml of water, 83.7 g (0.73 mol) of glutaric anhydride are added. The suspension is stirred for 1 hour, the resulting solution is evaporated to a volume of 150 ml, left in the refrigerator for 16 hours. The precipitate formed is filtered off, washed with 150 ml of methanol and dried. Purification is carried out by ion exchange chromatography on a Purolight resin in H ⁺ form, eluting with water. The fractions containing the target product are combined, evaporated before precipitation and left for 16 hours at + 4 ° C. The precipitate formed is filtered off, washed with 200 ml of methanol and dried to constant weight. Yield 98.8 g (55%). R _f 0.55 (1), 0.37 (2). _Mp = 222-224 ° C. [α] _D ²⁰ +15, 95 ° (C 0.53, water). [M + H] ⁺ 270.1. ¹ H-NMR spectrum (D ₂ O), δ, ppm: 1.60-1.80 (m, 2H, β-CH ₂ -Glt), 2.10-2.25 (m, 4H, α, γ-CH ₂ -Glt), 2.90-3.25 (m, 2H, β-CH ₂ -His), 4.40-4.50 (m, 1H, α-CH-His), 7 15 (s, 1H, CH-4-Im); 8.50 (s, 1H, CH-2-Im). Found,%: C 49.18; H 5.91; N, 15.42. C ₁₁ H ₁₅ N ₃ O ₅ . Calculated,%: C 49.07; H 5.62; N, 15.61.

Method B

To a suspension of 0.3 g (1.93 mmol) of histidine in 5 ml of water, 0.44 g (3.86 mmol) of glutaric anhydride dissolved in 2.5 ml of ethyl acetate are added with vigorous stirring. Stirred for 2 hours, adjusted to pH 7 with pyridine and stirred for another 1 hour. The ethyl acetate and water layer are separated. The aqueous layer was washed twice with ether, the ether layer was discarded. Water is removed in vacuo, the residue is dissolved in a minimum amount of water and ethanol is added until a white precipitate begins, left at + 4 ° C for 20 hours. The precipitate is filtered off and dried in vacuum. Yield 0.36 g (70%). R _f 0.56 (1), 0.35 (2). _Mp = 219-221 ° C. [α] _D ²⁰ = + 15.71 ° (C 0.56, water). [M + H] ⁺ 270.1. ¹ H-NMR spectrum (D ₂ O), δ, ppm: 1.40-1.55 (m, 2H, β-CH ₂ -Glt), 1.90-2.0 (m, 4H, α, γ-CH ₂ -Glt), 2.7-3.0 (m, 2H, β-CH ₂ -His), 4.20-4.30 (m, 1H, α-CH-His), 6 95 (s, 1H, 4-CH-Im), 8.30 (s, 1H, 2-CH-Im). HPLC under the conditions: (1) —individual peak, retention time 14.55 minutes. Found,%: C 49.07; H 5.65; N, 15.65. C ₁₁ H ₁₅ N ₃ O ₅ . Calculated,%: C 49.07; H 5.62; N, 15.61.

Example 2

N ^α -Succinyl-L-histidine (V)

The synthesis was carried out in accordance with method A described for compound IV.

Yield 0.08 g (57%).

R _f 0.44 (1), 0.25 (2).

_Mp = 179-181 ° C.

[α] _D ²⁰ = + 30.71 ° (C 0.56, water).

[M] ⁺ 255.2.

¹ H-NMR spectrum (D ₂ O), δ, ppm: 2.15-2.30 (m, 4H, (CH ₂ ) ₂ -Suc), 2.75-2.95 (m, 2H , β-CH ₂ -His), 4.25 (br s, 1H, α-CH-His), 6.95 (s, 1H, 4-CH-Jm), 8.25 (s, 1H, 2 -CH-Jm).

Found%: C, 47.09; H 5.04; N, 16.40. C ₁₀ H ₁₃ N ₃ O ₅ . Calculated%: C 47.06; H 5.13; N, 16.46.

The synthesis was carried out in accordance with method B described for compound IV.

Yield 0.101 g (67%).

R _f 0.45 (1), 0.27 (2).

_Mp = 178-180 ° C.

[α] _D ²⁰ = + 30.8 ° (C 0.57, water).

HPLC under conditions (1) —individual peak, retention time 7.54 min.

Found%: C 47.15; H 5.2; N, 16.50. C ₁₀ H ₁₃ N ₃ O ₅ . Calculated%: C 47.06; H 5.13; N, 16.46.

Example 3

N ^α -Glutaryl tryptophan (III)

To a suspension of 1.0 g (4.9 mmol) of tryptophan in 7 ml of water, a solution of 1 N NaOH (4.9 mmol) was added dropwise. To the resulting solution was added a solution of 0.56 g (4.9 mmol) of glutaric anhydride in 3 ml of ethyl acetate. The reaction mixture is stirred for 3 hours at room temperature in an argon atmosphere in the dark, left for 16 hours at + 4 °. The solvent from the reaction mixture was removed in vacuo. The resulting oily residue was dissolved in 30 ml of water with stirring, cooled to 0 °, a solution of 1 N HCl was added to pH 4. The product was extracted with ethyl acetate (3 × 25 ml). The combined ethyl acetate extract was cooled to 0 °, washed with water (4 × 25 ml) to pH 7, a solution of 5% HCl (5 ml), water (4 × 25 ml) to pH 7, dried over anhydrous Na ₂ SO ₄ for 1 hours. The precipitate of Na ₂ SO _{4 is} filtered off, washed with ethyl acetate, the solvent is removed in vacuo. A grayish solid residue is obtained, which is dried in vacuo.

Yield 1.0 g (70%).

R _f 0.54 (1).

_Mp = 150-152 ° C.

[α] _D ²⁰ = + 8.20 ° (C 0.5, methanol).

¹ H-NMR spectrum (CD ₃ OD), δ, ppm: 1.75-1.84 (m, 2H, β-CH ₂ -Glt), 2.15-2.30 (m, 4H, α, γ-CH ₂ -Glt), 3.30-3.40 (m, 2H, β-CH ₂ -Trp), 3.80-3.90 (m, 1H, α-CH-Trp), 6 97 (t, J = 7 Hz, 1H, CH-6-Ind), 7.06 (t, J = 7 Hz, 1H, CH-7-Ind), 7.15 (d, J = 7 Hz, 1H, CH-2-Ind), 7.33 (d, J = 7 Hz, 1H, CH-5-Ind), 7.55 (d, J = 7 Hz, 1H, CH-8-Ind).

HPLC under the conditions: (2) —individual peak, retention time 6.77 min.

Found,%: C 60.07; H 5.65; N, 8.75. C ₁₆ H ₁₈ N ₂ O ₅ Calculated,%: C 60.37; H 5.7; N, 8.8.

Example 4

N ^α -Succinyl-L-tryptophan (II)

The synthesis was carried out in accordance with the procedure described for compound III.

Yield 100.5 mg (67%).

R _f 0.63 (1).

[α] _D ²⁰ = + 21.05 ° (C 0.6, water).

¹ H-NMR spectrum (DMSO-d ₆ ), δ, ppm: 2.33-2.41 (m, 4H, α, β-CH ₂ -Suc), 2.93-3.01 (m , 1H, β-CH ₂ -Trp), 3.10-3.16 (m, 1H, β-CH ₂ -Trp), 4.39-4.47 (m, 1H, α-CH-Trp), 6.93-7.06 (m, 2H, CH-6.7-Ind), 7.11 (d, J 2.2 Hz, 1H, CH-2-Ind), 7.30-7.32 ( m, 1H, CH-5-Ind), 7.44-7.47 (m, 1H, CH-8-Ind). [M] ⁺ 304.3.

HPLC under the conditions: (2) —individual peak, retention time 6.35 minutes.

Found,%: C 59.07; H 5.65; N, 9.35. C ₁₅ H ₁₆ N ₂ O ₅ Calculated,%: C 59.21; H 5.3; N, 9.21.

Example 5

Monosodium salt N ^α -glutaryl-L-histidine (IV)

To a solution of 1.0 g (3.7 mmol) of N ^α -glutaryl-L-histidine in 15 ml of water, a solution of 0.15 g (3.7 mmol) of NaOH in 20 ml of water is added with stirring and cooling to + 5 ° C. . The solution was stirred for 30 minutes, the solvent was removed in vacuo. Benzene was added in portions to the oily residue, and the solvent was removed in vacuo. The solid residue is dried over granular alkali.

Yield 1.07 g (99.7%).

_Mp = 208-210 ° C.

[α] _D ²⁰ = + 16.27 ° (C 0.58, water).

Found,%: C 45.25; H 5.51; N, 14.52. C ₁₁ H ₁₅ N ₃ O ₅ Na. Calculated,%: C 45.21; H 5.17; N, 14.38.

Example 6

Monosodium salt N ^α -succinyl-L-histidine (V)

The synthesis was carried out in accordance with the procedure described for the monosodium salt of N ^α -glutaryl-L-histidine (IV) (example 5).

Yield 1.06 g (97.0%).

[α] _D ²⁰ = + 40.21 ° (C 0.48, water).

Found,%: C 43.25; H 4.51; N, 15.52. C ₁₀ H ₁₃ N ₃ O ₅ Na. Calculated,%: C 43.17; H 4.71; N, 15.10.

Example 7

Monosodium salt N ^α -succinyl-L-tryptophan (II)

The synthesis was carried out in accordance with the procedure described for the monosodium salt of N ^α -glutaryl-L-histidine (IV) (example 5).

Yield 0.21 g (98.0%).

_Mp = 147-150 ° C.

[α] _D ²⁰ = + 22.02 ° (C 0.39, water).

Found,%: C 55.25; H 4.51; N, 8.32. C ₁₅ H ₁₆ N ₂ O ₅ Na. Calculated,%: C 55.05; H 4.93; N, 8.56.

HPLC under the conditions: (2) —individual peak, retention time 6.56 min.

Example 8

Monosodium salt N ^α -glutaryl-L-tryptophan (III)

The synthesis was carried out in accordance with the procedure described for the monosodium salt of N ^α -glutaryl-L-histidine (IV) (example 5).

Yield 0.11 g (99.0%).

_Mp = 128-130 ° C.

[α] _D ²⁰ = + 22.06 ° (C 0.34, methanol).

Found,%: C 56.15; H 5.21; N, 8.22. C ₁₆ H ₁₈ N ₂ O ₅ Na. Calculated,%: C 56.30; H 5.32; N, 8.21.

HPLC under the conditions: (2) —individual peak, retention time 6.96 min.

Example 9

Disodium salt N ^α -glutaryl-L-histidine (IV)

To a solution of 1.0 g (3.7 mmol) of N ^α -glutaryl-L-histidine in 15 ml of water, a solution of 0.3 g (7.44 mmol) of NaOH in 15 ml of water is added with stirring and cooling to + 5 ° C. . The solution was stirred for 30 minutes, the solvent was removed in vacuo. Benzene was added in portions to the oily residue, and the solvent was removed in vacuo. The solid residue is dried over granular alkali.

Yield 1.15 g (99.0%).

[α] _D ²⁰ = + 11.92 ° (C 0.57, water).

Found,%: C 41.25; H 4.51; N, 13.52. C ₁₁ H ₁₅ N ₃ O ₅ Na ₂ . Calculated,%: C 41.91; H 4.80; N 13.3.

Example 10

Disodium salt N ^α -succinyl-L-histidine (V)

The synthesis was carried out in accordance with the procedure described for the disodium salt of N ^α -glutaryl-L-histidine (IV) (example 9).

Yield 1.16 g (99.0%).

_Mp = 124-128 ° C.

[α] _D ²⁰ = + 20.06 ° (C 0.67, water).

Found,%: C 39.55; H 4.31; N, 13.52. C ₁₀ H ₁₃ N ₃ O ₅ Na ₂ . Calculated,%: C 39.88; H 4.35; N, 13.95.

Example 11

Disodium salt N ^α -succinyl-L-tryptophan (II)

The synthesis was carried out in accordance with the procedure described for the disodium salt of N ^α -glutaryl-L-histidine (IV) (example 9).

Yield 0.56 g (97.7%).

Found,%: C 51.35; H 4.31; N, 8.22. C ₁₅ H ₁₆ N ₂ O ₅ Na ₂ . Calculated,%: C 51.43; H 4.60; N, 8.0.

Example 12

Disodium salt N ^α -glutaryl-L-tryptophan (III)

The synthesis was carried out in accordance with the procedure described for the disodium salt of N ^α -glutaryl-L-histidine (IV) (example 9).

Yield 0.56 g (98.5%).

Found,%: C 52.55; H 4.71; N, 7.52. C ₁₆ H ₁₈ N ₂ O ₅ Na ₂ . Calculated,%: C 52.75; H 4.98; N, 7.69.

Biological Activity Tests

Example 13

The effect of compounds of General formula I on allergic reactions of the immediate type (test of ovalbumin-induced (OA) degranulation of blood basophils of an immunized guinea pig in vitro)

The selection of leukocytes from the blood of guinea pigs was carried out according to the method of Friemel (Immunological methods. / Ed. G. Fremel / - M .: Medicine, 1987, p. 222 in our modification).

For the test, guinea pigs of both sexes weighing 600-800 g were used. Animals were immunized once with a mixture of ovalbumin 10 μg and 100 mg of aluminum hydroxide per animal according to Andersson (Anderson P., Antigen-induced bronchial anaphulaxis in actively sensitized geinea-pigs. // Allergy 1980, Vol. 35, p. 63-71).

Under ether anesthesia, 15 ml of blood was taken from the heart of a guinea pig. For the isolation of basophils in the leukocyte suspension, double cell deposition was used - by means of EDTA and using a citrate-containing precipitating liquid.

The blood was mixed with a 5% solution of EDTA · Na ₂ 2H ₂ O (Sigma) in a ratio of 9: 1 and after 30 minutes it was gently centrifuged (12 minutes at 80 g). The supernatant was collected and centrifuged for 15 min at 500 g.

A citrate-containing precipitating liquid (3) was added to the remaining blood cells in a proportion of 3:10 (thermostatted at 37 ° C for 30 min). The supernatant fraction enriched in leukocytes was centrifuged for 7 min at 100 g. A 0.85% NaCl solution was added to the leukocyte pellet, and the cell concentration was adjusted to 30 × 10 ³ / μl.

Testing of basophil degranulation in vitr o [Reference for clinical laboratory research methods. / Ed. E.A. Kost / - M .: Medicine, 1975, p. 130].

To place the test in a centrifuge tube (3 tubes are used for each sample), 300 μl of cell suspension were placed, then 300 μl of saline solution of the test compound (or saline in the control of spontaneous and maximum degranulation) was added and incubated at 37 ° C for 15 min , then 300 μl of a 1% OA saline solution was added to each tube (the same amount was added to the spontaneous degranulation control) and reincubated at 37 ° С for 10 min. The working concentration of leukocytes is 10 ⁴ / μl. Samples (100 μl) were taken from each tube into separate tubes to assess the complete degranulation of basophils, and chilled saline (5 ml per tube) was added to the remaining cells to stop the degranulation reaction, then centrifuged for 7 min at 100 g, and from the pellet prepared preparations for microscopy. Fixation and staining of the preparations was carried out according to the method of Seder et al. (Seder RA et al., Mouse splenic and bone marrow cell populations that express high - affinity FCε receptors and produce interleukin-4 are highly enriched in basophils. // Proc. Natl. Acad. USA, 1991, V.88, p. 2835-2839).

To identify the specific granularity of basophils, dye 0.5% alcian blue (pH 1.0) was used, the nuclei were stained with safranin (0.1% solution in 1% acetic acid). The drugs were used to assess the total inhibition of degranulation

Inhibition of total degranulation (TG) (%) was calculated by the formula

, где

where

max -% degranulated basophils with maximum degranulation (OA),

spont. -% degranulated basophils during spontaneous degranulation (control),

expert. -% degranulated basophils after exposure to the test compound.

Assess complete basophil degranulation

Samples taken after setting the basophil degranulation test (100 μl) were placed in dye tubes (0.5% alcian blue, pH 1.0) in a 1: 1 ratio. Staining was performed at room temperature for at least 50 minutes. The number of stained basophils was counted using a Fuchs-Rosenthal camera. Inhibition of complete degranulation (TPD) of basophils was calculated by the formula:

TPD (%) = 1 - [(M cf (k) - M cf (exp.)] / [M cf (k) - M cf (OA)] × 100, where

M cf (k) - average (for 3 samples) the number of basophils in the test of spontaneous degranulation;

M cf (OA) - average (for 3 samples) the number of basophils in the test of maximum antigen-induced degranulation;

M cf (exp) is the average (for 3 samples) the number of basophils in the degranulation test after incubation with the test compound.

table 2
Inhibition of OA-induced degranulation of blood basophils of immunized guinea pigs in vitro under the influence of compounds of the general formula I Experience No. Groups Inhibition of complete degranulation (TPD), (%) The number of fully degranulated basophils, (%) one. Control 1 (spontaneous degranulation) one hundred 0 2. Ovalbumin 1%
(max degranulation) 0 35.2 ± 0.8 3. Compound IV
(10 ^-3 M) 99.2 ± 11.2 2.6 ± 2.6 * four. Compound IV
(10 ^-4 M) 99.5 ± 12.0 2.9 ± 1.6 * 5. Compound IV
(10 ^-5 M) 113.5 ± 2.7 0 6. Compound IV
(10 ^-6 M) 90.0 ± 1.8 3.9 ± 0.6 7. Compound IV
(10 ^-7 M) 76.7 ± 1.5 9.1 ± 0.8 8. Glutaryl histamine
(10 ^-3 M) 9.3 ± 5.5 31.6 ± 6.33 9. Glutaryl histamine
(10 ^-4 M) 24.1 ± 1.1 25.3 ± 3.38 10. Glutaryl histamine
(10 ^-5 M) 0 29.8 ± 6.73 eleven. Control 2 one hundred 0 12. Ovalbumin 1%
(max degranulation) 0 38.9 ± 8.43 13. Compound V
(10 ^-3 M) 10.6 ± 7.6 35.3 ± 7.29 fourteen. Compound V
(10 ^-4 M) 18.9 ± 11.8 31.2 ± 6.8 fifteen. Compound V
(10 ^-5 M) 44.0 ± 11.27 24.6 ± 10.38 16. Hydrocortisone
(10 ^-3 M) 71.0 ± 1.6 10.0 ± 0.7 17. Hydrocortisone
(10 ^-4 M) 48.0 ± 0.8 17.3 ± 0.9 eighteen. Hydrocortisone
(10 ^-5 M) 40.0 ± 0.6 20.0 ± 1.3 (* - P <0.001)

The data in Table 2 show that, compared with glutaryl histamine, compound (IV) has a pronounced anti-anaphylactic effect, which is manifested by almost 100% inhibition of degranulation in the test of complete OA-induced degranulation of blood basophils of actively immunized guinea pigs (in vitro anaphylaxis reaction in calcium-free medium). A significant anti-anaphylactic effect of compound IV is also manifested in a decrease in the number of degranulated cells, especially expressed in a concentration of 10 ^-5 M (absence of degranulated cells).

Example 14

The study of the effect of compounds of General formula I on systemic anaphylaxis in vivo

We used a model of bronchospasm in non-narcotic actively sensitized guinea pigs with aerosol exposure to ovalbumin as an antigen (Kovaleva V.L. “Methodological guidelines for the study of bronchodilators, mucolytic and anti-inflammatory drugs” .// Guide to the experimental (preclinical) study of new pharmacological substances, Moscow, 2000, p. 242-250).

Guinea pigs were sensitized with ovalbumin according to the Andersson method (Anderson P., Antigen-induced bronchial anaphulaxis in actively sensitized geinea-pigs.//Allergy, 1980, Vol. 35, p. 63-71) and bronchospasm was induced 1-2 months after sensitization aerosol administration of a permissive dose of ovalbumin (3 mg / kg in 1 ml of saline).

In the experimental groups, the test compounds were administered intragastrically to guinea pigs for three days at a dose of 10 μg / kg, 50 μg / kg and 150 μg / kg using a probe. In another series of experiments, the studied compounds at a dose of 50 μg / kg (in 1 ml of saline) were administered by inhalation (using a nebulizer) also for three days once a day. The control group was injected with saline. Ovalbumin was inhaled with a nebulizer 1 hour after the last injection of the substances and the duration (in seconds) and the intensity of the bronchospastic reaction of the animals were evaluated.

Table 3
Inhibition of the systemic anaphylactic reaction of guinea pigs with the inhalation of compound IV at a dose of 50 μg / kg Groups The duration of the acute phase, sec The duration of the subacute phase, sec Control 1
(saline) 180 ± 6 650 ± 34 Compound IV
50 mcg / kg 0 400 ± 25

Table 4
Inhibition of the systemic anaphylactic reaction of guinea pigs with intragastric administration of compound IV at doses of 10 and 150 μg / kg (M ± m) Groups The duration of the acute phase, sec Total reaction time, sec Control 2
(saline) 296.7 ± 104.6 628.3 ± 80.6 Compound IV
10 mcg / kg 68.0 ± 54.7 * 428.0 ± 75.0 Compound IV
150 mcg / kg 72.0 ± 42.1 * 337.0 ± 78.5 * * - P <0.001

The experimental results presented in tables 3 and 4 show that compound IV, when administered intragastrically at doses of 10 and 150 μg / kg and inhaled at a dose of 50 μg / kg, showed antianaphylactic activity. Inhaled administration of a substance at a dose of 50 μg / kg blocked the development of the acute phase of the bronchoconstrictor reaction, which, causing suffocation, causes the death of animals. With the intragastric administration of compound IV at doses of 10 μg / kg and 150 μg / kg, a significant protective effect was revealed with respect to antigen-induced bronchospasm.

Thus, compound IV exhibits a significant protective effect on the in vivo systemic anaphylactic reaction.

Example 15

Antiallergic effect of compounds of the general formula I on the model of allergic rhinitis in guinea pigs

The model of allergic rhinitis in guinea pigs was used.

Guinea pigs were immunized according to a certain scheme for 1.5-2 months (Hutson PA, Church MK et al., 1988): at first, animals were immunized with intraperitoneal administration of ovalbumin at a dose of 10 mg / kg with a 7-day interval (twice), then, the pigs were inhaled using a nebulizing technique (Pari) with an ovalbumin solution in increasing concentration, starting from 0.1% to 1% with an interval of 4 days between inhalations. The last dose of ovalbumin was introduced into the nasal passages using a micropipette. 24 hours after the last administration of ovalbumin, a nasal wash was taken (through a system of special tubes) and changes in the nasal mucosa were evaluated using a complex of methods: histological and cytological. The test compounds (0.1% solution) were administered daily as inhalations using a nebulizer technique for 6 days, and antigen provocation (OA) was provoked on the 6th day of administration. A nasal wash was obtained one day after the provocation.

Table 5
The effect of compound IV on cytosis (absolute number of cells in 1 μl) in nasal washout Group Intact control Model + provocation (rhinitis) Compound IV
(0.1% solution) N 6 7 7 Cytosis 18.0 ± 2.10 67.2 ± 6.47 ** 43.7 ± 6.65 ° * * - difference from intact control; ° - difference from the model
(° - P <0.05; * - P <0.01; ** - P <0.001)

Table 6
The effect of compound IV on the cytogram (%) of the nasal wash of guinea pigs Group
Subpopulation Intact control
n = 6 Model
(rhinitis)
n = 7 Compound IV
(0.1% solution)
n = 7 Macrophages 21.8 ± 1.64 12.7 * ± 1.57 14.6 * ± 1.27 Lymphocytes 4.7 ± 0.88 6.1 ± 1.42 5.7 ± 1.54 Neutrophils 0.3 ± 0.21 3.3 * ± 0.80 7.8 ** ° ± 1.35 Eosinophils 7.0 ± 2.45 47.9 *** ± 5.37 6.9 °°° ± 1.32 Epithelial cells 66.2 ± 3.09 27.1 *** ± 5.01 63.6 °°° ± 3.02 * - difference from intact control; ° - difference from the model (rhinitis)
(° - P <0.05; **, °° - P <0.01; ***, °°° - P <0.001)

Table 7
The effect of compound IV on the absolute number of cell subpopulations of nasal washout of guinea pigs (in 1 μl) Group
Subpopulation Intact control
n = 6 Model
(rhinitis)
n = 7 Compound IV
(0.1% solution)
n = 7 Macrophages 6.2 ± 1.29 7.9 ± 1.88 6.1 ± 0.75 Lymphocytes 0.9 ± 0.24 4.1 * ± 1.16 3.2 * ± 52 Neutrophils 0,03 ± 0,021 2.1 ± 0.67 3.1 ± 1.02 Eosinophils 1.2 ± 0.46 26.5 *** ± 4.47 3.2 °° ± 0.97 Epithelial cells 11.8 ± 1.4 11.3 ± 2.10 26.3 ** °° ± 4.31 Note: * - difference from intact control; ° - difference from the model
(* - P <0.05; **, °° - P <0.01; *** -P <0.001)

From the data of tables 5-7 it follows that in the conditions of modeling allergic rhinitis, compound IV significantly suppresses eosinophilic inflammation. This is confirmed by a decrease in the norm of the absolute and relative number of eosinophils, as well as a significant decrease in cytosis in the nasal wash.

Example 16

Antiallergic activity of compounds of the general formula I in a model of allergic pneumonia in guinea pigs

A model of allergic pneumonia in guinea pigs was used.

Immunization of animals is similar to that described in example 14.

24 hours after the last administration of ovalbumin, a bronchoalveolar lavage was taken (via a cannula inserted into the trachea), and changes in the bronchial mucosa were evaluated using a complex of methods: histological and cytological.

The test compounds (0.1% r-p) were administered daily as inhalations using a nebulizer technique for 6 days, and antigen provocation (OA) was provoked on the 6th day of administration.

Table 8
The effect of compound IV on cytosis (absolute number of cells in 1 μl) in bronchoalveolar washout Group Intact control Model + provocative. (rhinitis) Compound IV
(0.1% solution) N 6 5 7 Cytosis 726.8 ± 82.4 1849.4 ± 287.3 * 1331.3 ± 277.4 * P <0.01 - difference from intact control

Table 9
The effect of compound IV on the absolute number of cell subpopulations of bronchoalveolar washout of guinea pigs (in 1 μl) Group
Subpopulation Intact control Model Compound IV
(0.1% solution) Macrophages 502.6 ± 60.31
(n = 6) 680.3 ± 105.0
(n = 5) 640.1 ± 57.98
(n = 6) Lymphocytes 101.4 ± 24.3
(n = 6) 328.3 ** ± 49.18
(n = 5) 233.0 * ± 45.77
(n = 7) Neutrophils 0
(n = 6) 56.8 ± 17.08
(n = 5) 16.9 ° ± 4.22
(n = 7) Eosinophils 37.0 ± 9.04
(n = 5) 773.0 ** ± 171.7
(n = 5) 487.4 ** ± 98.70
(n = 6) Epithelial cells 16.9 ± 6.09
(n = 6) 0
(n = 5) 2.48 * ± 1.84
(n = 7) * - difference from intact control; ° - difference from the model
(*, ° - P <0.05; ** - P <0.01)

From the data of tables 8 and 9 it follows that compound IV in the conditions of the model of allergic pneumonia significantly inhibits the inflammatory process, which is manifested by a decrease in cytosis, a decrease in the content of the key cell of allergic inflammation - eosinophils, a sharp decrease in neutrophils, and also a decrease in the number of lymphocytes.

Example 17

The study of the anti-inflammatory effect of compounds of the general formula I on a model of pneumonia in rats induced by Sephadex

Rat Sephadex-Induced (6-Day) Pneumonia

In the experiments, male Wistar rats weighing 270-300 g were used.

Inflammation in the lungs was induced by a single inhalation administration of Sephadex A-25 (hydrophilic powder with particle sizes from 20 to 80 μm) at a dose of 5 mg / kg using a dosing device that is a laboratory analogue of the Cyclochaler inhaler (Research Institute of Pulmonology of the Russian Federation).

The method of inhalation administration of Sephadex and pharmacological substances

Rats using an original dosing device for inhalation of dry powders under ether anesthesia were administered Sephadex A-25 at a dose of 5 mg per 1 kg of body weight. After the introduction of Sephadex A-25, Wistar rats quickly emerged from anesthesia and apparently did not have any behavioral or pattern of respiration. Substances in the form of a dry powder were administered by inhalation at a dose of 500 μg / kg 1 hour after the administration of Sephadex, then for 5 consecutive days daily 1 time per day in the same morning hours. The control is represented by 2 groups: a group of intact animals and a group of rats to which Sephadex was once inhaled.

The results of the therapeutic effect of pharmacological substances on the development of the inflammatory process in the lungs were evaluated 6 days after aerosol exposure to Sephadex using morphological and morphometric indicators (bulk density of emphysema and alveolitis).

Methods used in the work

Histological

A histological examination of the lungs stained with hematoxylin and eosin was performed.

Morphometric

Histological sections of the lungs with a thickness of 4-5 μm were prepared, in which the number of neutrophils of the interalveolar septa was counted, and the bulk density of alveolitis and emphysema was estimated using the Avtandilov grid (Avtandilov G.G. Introduction to quantitative pathological morphology. // - M .: Medicine, 1980, p. 203). A morphometric study of the lymphoid tissue of the lungs was also performed. For this purpose, lung macroscopic preparations were fixed according to the method of Beinenstock et al. (Bienenstock J., Johnson N., Perey D.Y.E., Bronchial lymphoid tissue 1. Morphologic characteristics // Lab. Invest., 1973, v. 28, p. 693-698). Lungs with trachea were removed from the chest cavity, and the macro was placed in a 2% aqueous solution of acetic acid. After 18-24 hours, the trachea, the main and lobar bronchi were dissected; Using the spot counting method under a magnifying glass (× 7), a morphometric assessment of the bulk density of lymphoid tissue associated with the bronchi was performed. The volumetric density of alveolitis and emphysema was determined by the method of point counting.

Cytological

Bronchoalveolar lavage in rats and guinea pigs was obtained under hexenal anesthesia by twice washing the lungs through the trachea with 10 ml of physiological saline. Cell viability was determined in a trypan blue test. In a liquid of bronchoalveolar washout (ALS), the absolute number of cells in 1 ml (cytosis) was determined using a Goryaev camera. In smears from a sediment of ALS fluid obtained by centrifugation at 200 g for 10 minutes and then stained according to Romanovsky-Giemsa, the endopulmonary cytogram was calculated (in percent) [Avtsyn A.P., Lukomsky G.I., Romanova L.K. . et al. Endopulmonary cytogram. // Owls. honey., 1982, No. 7, p. 8-14].

The research results were processed by methods of variation statistics using t-student criterion.

Table 10
Cytosis indices of bronchoalveolar washout of Wistar rats after aerosol exposure to Sephadex A-25 and treatment with pharmacological agents (M ± m)
Cytosis
The absolute number of cells in 1 μl of BAL Criterion Intact
n = 5 Sephadex
(model)
n = 6 Compound IV
n = 6 Cytosis 160.4 ± 20.65 259.2 * ± 32.42 178.6 * ± 20.4 R 0.05 0.05 Note: * - difference from intact control

Table 11
Indicators of the cellular composition of bronchoalveolar washout of Wistar rats after aerosol exposure to Sephadex A-25 and treatment of the test compound (M ± m)
Cytosis
The absolute number of cells of various subpopulations in 1 μl of BAL Subpopulation Intact
the control Model
(Sephadex) Compound IV N four 5 6 Macrophages 124.8 ± 16.35 226.1 * ± 30.83 152.4 ± 18.5 Lymphocytes 15.5 ± 4.27 28.2 ± 6.01 22.0 * ± 3.5 Neutrophils 0 20.4 * ± 6.38 5.1 * ± 0.6 Note: * - difference from intact control (P <0.05)

Histological examination of the lungs

Compound IV caused a distinct anti-inflammatory effect: the prevalence of alveolitis was significantly lower compared to the model group of animals; emphysema was practically not detected; infiltration of the interalveolar septa by neutrophils was not noted. By the level of cytosis and the number of neutrophils in BAL, the inflammatory process is also significantly less pronounced than in animals treated with Sephadex for 5 days.

Thus, the whole complex of experimental models used indicates a significant anti-allergic, anti-anaphylactic and anti-inflammatory activity of compounds of the general formula I, which is manifested both in in vitro tests and in modeling of allergic and inflammatory pathology in vivo.

Example 18

The study of the lipid-lowering effect of the compounds of the general formula (I) of the rat hypercholesterolemia model

The study was conducted on male Wistar rats weighing 200 ± 20 g. Hyperlipidemia was caused by oral administration of a cholesterol load - an oily suspension of cholesterol:

olive oil (Acorsa, Spain) - 5 ml / kg of animal weight,

cholesterol (Sigma, USA) - 1 g / kg of weight,

sodium cholate (Sigma, USA) - 100 mg / kg of weight.

As a comparison drug, a drug from the statin group was used - mevacor (lovastatin) from Merck Sharp & Dohn at a dose of 40 mg / kg. A cholesterol suspension was administered in the morning daily for 10 days. The test compounds (at a dose of 500 μg / kg) and the reference drug (at a dose of 40 mg / kg) were administered to animals along with a cholesterol suspension for 10 days. All animals received standard briquetted food.

Animals were divided into the following groups:

"Control" - intact animals (n = 6),

"Cholesterol" - rats receiving per os cholesterol load (n = 10),

"Lovastatin" - rats receiving per os cholesterol load and lovastatin (n = 10),

"Compound IV" - rats, received per os cholesterol load and the test compound IV (n = 10).

A blood sample was taken on the 5th, 8th, 10th day of the experiment.

Statistical processing of the lipid-lowering effect of the test substances was carried out with respect to the “cholesterol” group (table 13).

Table 12
The content of cholesterol and triglycerides in the blood serum and liver of rats receiving olive oil, cholesterol and compound IV simultaneously with cholesterol load for 10 days per os The control
(n = 6) Cholesterol
(n = 10) Lovastatin
(n = 10) compound IV
(n = 9) Total cholesterol Serum (mg / 100 ml) 67.2 ± 6.1 120.0 ± 8.4 100.5 ± 6.7 * 96.6 ± 5.7 ** HDL
(mg / 100 ml) 41.6 ± 1.3 54.3 ± 1.7 50.6 ± 1.3 50.5 ± 1.3 * LDL + VLDL (mg / 100 ml) 25.6 ± 0.8 65.7 ± 1.2 49.9 ± 0.9 *** 46.1 ± 0.9 *** Liver
(mg / g tissue) 2.29 ± 0.13 3.6 ± 0.4 2.41 ± 0.17 *** 2.78 ± 0.28 *** Triglycerides Serum (mg / 100 ml) 85.7 ± 9.2 99.2 ± 8.7 93.6 ± 7.5 95.1 ± 7.9 Liver
(mg / g tissue) 3.89 ± 0.14 6.83 ± 0.39 6.25 ± 0.13 5.53 ± 0.19 *** * - p <0.1
** - p <0.05
*** - p <0.01

The introduction of compound IV at a dose of 500 μg / kg led to a significant decrease in total serum cholesterol by 19.5%, liver by 22.7%, LDL cholesterol by 29.8%, and liver triglycerides by 19%. The glutaryl histamine compound, reducing total cholesterol by only 9%, had an effect only on LDL and VLDL (low and very low density lipoproteins), as shown in the publication of international application WO 99/01103, and, obviously, less effective than compound IV. in a similar biological experiment.

The following are the results of studies of other claimed compounds.

The experimental groups included:

1) "Cholesterol" - received within 10 days per os an oil suspension of cholesterol,

2) "compound IV" - rats, received per os oil suspension of cholesterol and test compound IV,

3) "compound IV - 1Na" - rats, received per os oil suspension of cholesterol and the monosodium salt of the compound IV,

4) "compound IV - 2Na" - rats, received per os oil suspension of cholesterol and the test disodium salt of compound IV,

5) "compound V - 1Na" - rats, received per os oil suspension of cholesterol and the monosodium salt of the compound V,

6) "compound III - 1Na" - rats, received per os oil suspension of cholesterol and the monosodium salt of compound III,

7) "compound II - 1Na" - rats, received per os oil suspension of cholesterol and the investigated monosodium salt of compound II,

8) "Sim" - rats receiving per os an oily suspension of cholesterol and simvastatin,

9) control - intact rats before the experiment.

On the 10th day of the experiment, blood samples were taken after decapitation of the animals. Before decapitation, the animals starved for 12 hours.

Serum levels of total cholesterol, triglycerides, high density lipoprotein cholesterol (HDL) were measured. Low and very low density lipoprotein cholesterol was calculated by the difference between total cholesterol and HDL cholesterol.

The content of total cholesterol and triglycerides in blood serum was determined by the enzymatic method.

Determination of the cholesterol content in high density lipoproteins (α-LP) was carried out by the method of deposition of LDL and VLDL with phosphoric tungsten acid and magnesium ions.

Statistical processing

The data in the tables are presented as mean ± standard error of the mean. The significance of differences between the “cholesterol” and “drug ...” groups was evaluated using the two-sample student t-test. The probability of error (p) is indicated in the columns of the table.

Data on the effect of the studied compounds on the cholesterol and triglycerides in the blood serum of rats receiving cholesterol load are presented in tables 13-20.

Table 13
Experimental Hypercholesterolemia Rat lipid metabolism (mg / 100 ml) before the experiment (n = 50) 10 days of cholesterol load (n = 20) Total cholesterol Serum 59.9 ± 1.4 135.7 ± 10.9 HDL 38.2 ± 1.2 32.5 ± 1.1 LDL + VLDL 20.3 ± 1.3 103.2 ± 11.5 Triglycerides Serum 83.6 ± 4.6 156.6 ± 11.4 HDL 19.0 ± 1.4 24.3 ± 1.8 LDL + VLDL 58.7 ± 6.2 132.6 ± 12.8

A ten-day administration to rats of an oily suspension of cholesterol led to a significant increase in the content of total cholesterol in serum by 2.3 times, triglycerides by 1.9 times. With the development of induced hyperlipidemia, HDL cholesterol decreased by 15%. LDL + VLDL cholesterol increased 5 times. LDL + VLDL triglycerides increased 2.3 times.

Table 14
The content of cholesterol and triglycerides (mg / 100 ml) in rat blood serum on the 5th day of the experiment Indicators Groups "Cholesterol" (n = 20) "Compound IV"
(n = 20) "Compound IV-1Na"
(n = 20) "Compound IV-2Na"
(n = 19) "Sim"
(n = 10) Total serum cholesterol 92.3 ± 3.5 84.2 ± 3.9 72.0 ± 3.0
p = 0.0001 80.6 ± 2.8
p = 0.013 73.6 ± 7.3
p = 0.04 HDL 34.3 ± 1.3 35.8 ± 1.2 33.1 ± 1.4 35.0 ± 1.3 27.8 ± 2.7 LDL + VLDL 58.0 ± 4.1 48.5 ± 4.5 38.8 ± 2.9
p = 0,0006 44.3 ± 2.7
p = 0.02 43.0 ± 8.3 Serum Triglycerides 110.8 ± 7.1 120.6 ± 8.9 106.6 ± 6.2 88.3 ± 4.8
p = 0.013 108.9 ± 6.9

Table 15
The content of cholesterol and triglycerides (mg / 100 ml) in rat blood serum on the 8th day of the experiment Indicators Groups "Cholesterol"
(n = 20) "Compound IV"
(n = 20) "Compound IV-1Na"
(n = 20) "Compound IV-2Na"
(n = 19) "Sim"
(n = 10) Total serum cholesterol 127.2 ± 10.6 103.3 ± 8.1
p = 0.08 99.1 ± 6.0
p = 0.03 90.2 ± 5.1
p = 0.004 100.1 ± 12.8 HDL 30.2 ± 1.0 35.9 ± 1.2
p = 0,0007 37.1 ± 1.4
p = 0,0003 35.9 ± 1.8
p = 0.01 33.2 ± 1.8 LDL + VLDL 97.0 ± 11.3 67.3 ± 8.7
p = 0.04 61.9 ± 6.6
p = 0.01 54.3 ± 5.8
p = 0.002 66.9 ± 13.4
p = 0.1 Triglycerides Serum 150.6 ± 13.6 154.2 ± 11.4 146.4 ± 11.2 125.8 ± 9.4 119.9 ± 10.5
p = 0.085

Table 16
The content of cholesterol and triglycerides (mg / 100 ml) in rat serum on the 10th day of the experiment Indicators Groups "Cholesterol"
(n = 20) "Compound IV"
(n = 20) "Compound IV-1Na"
(n = 20) "Compound IV-2Na"
(n = 19) "Sim"
(n = 10) Total serum cholesterol 135.7 ± 10.9 95.2 ± 5.4
p = 0.003 97.4 ± 7.1
p = 0.006 97.9 ± 3.9
p = 0.003 93.9 ± 10.2
p = 0.01 HDL 32.5 ± 1.1 36.5 ± 0.9
p = 0.093 37.7 ± 1.0
p = 0.02 40.0 ± 1.9
p = 0.002 32.7 ± 1.2 LDL + VLDL 103.2 ± 11.5 58.7 ± 5.3
p = 0.002 59.7 ± 7.2
p = 0.003 57.3 ± 4.3
p = 0.002 61.2 ± 9.7
p = 0.01 Serum Triglycerides 156.6 ± 11.4 143.3 ± 8.9 132.5 ± 8.4
p = 0,096 127.3 ± 6.2
p = 0.03 142.0 ± 5.4

Table 17
The development of hypercholesterolemia in experimental animals in the group "Cholesterol" Rat lipid metabolism (mg / 100 ml) * before the experiment
(n = 30) 5 days of cholesterol load
(n = 12) 8 days of cholesterol load
(n = 12) 10 days of cholesterol load
(n = 12) Total cholesterol Serum 89.1 ± 1.8 112.9 ± 9.2
p = 0.025 153.0 ± 14.7 144.6 ± 12.8 HDL 66.7 ± 1.1 47.5 ± 3.7
p <0.02 50.3 ± 5.1 46.7 ± 2.9 LDL + VLDL 22.5 ± 1.7 65.4 ± 10.6
p <0.001 100.9 ± 17.3 97.9 ± 14.0 Triglycerides Serum 74.8 ± 4.1 94.5 ± 9.2 129.7 ± 17.9 115.8 ± 18.9 HDL 43.1 ± 1.9 35.0 ± 3.1
p <0.05 46.0 ± 4.6 35.5 ± 3.1 LDL + VLDL 31.7 ± 3.7 59.5 ± 7.6
p <0.05 76.0 ± 14.7 80.3 ± 16.2

A ten-day administration to rats of an oily suspension of cholesterol led to a significant increase in serum total cholesterol 1.7 times, triglycerides 1.6 times (table 17). HDL cholesterol decreased by 28% from the initial 66.7 to 48 mg / 100 ml with the development of induced hyperlipidemia. LDL cholesterol + VLDL increased 4.4 times from 22.5 to 99 mg / 100 ml.

Table 18
The content of cholesterol and triglycerides (mg / 100 ml) in rat serum on the 5th day of the experiment Indicators Groups "Cholesterol"
(n = 12) "Compound IV-1Na"
(n = 12) "Connection V-1Na"
(n = 12) "Compound III-1Na"
(n = 12) " Compound II-1Na "
(n = 12) Total serum cholesterol 112.9 ± 9.2 94.4 ± 3.4
p = 0.079 96.9 ± 4.5 89.0 ± 5.0
p = 0.035 109.7 ± 9.4 HDL 47.5 ± 3.7 60.7 ± 3.1
p = 0.012 55.1 ± 2.8
p = 0.11 49.0 ± 2.1 49.5 ± 3.8 LDL + VLDL 65.4 ± 10.6 33.7 ± 4.5
p = 0.015 41.7 ± 6.5
p = 0,073 40.0 ± 5.4
p = 0.048 60.2 ± 10.6 Triglycerides Serum 94.5 ± 9.2 83.2 ± 6.3 63.3 ± 5.4
p = 0.009 87.4 ± 9.4 80.9 ± 5.5 HDL 35.0 ± 3.1 39.4 ± 3.5 30.9 ± 1.8 30.0 ± 2.2 32.6 ± 2.5 LDL + VLDL 59.5 ± 7.6 43.7 ± 4.6
p = 0.09 32.4 ± 4.4
p = 0.007 57.4 ± 9.1 48.2 ± 4.9

Table 19
The content of cholesterol and triglycerides (mg / 100 ml) in rat serum on the 8th day of the experiment Indicators Groups "Cholesterol"
(n = 12) "Compound IV-1Na"
(n = 12) "Connection V-1Na"
(n = 12) "Compound III-1Na"
(n = 12) " Compound II-1Na"
(n = 12) Total cholesterol Serum 153.0 ± 14.7 120.8 ± 4.3
p = 0.07 124.8 ± 5.8
p = 0.11 108.8 ± 8.2
p = 0.03 140.8 ± 13.6 HDL 50.3 ± 5.1 54.9 ± 2.4 54.2 ± 3.4 48.4 ± 2.7 52.9 ± 4.1 LDL + VLDL 100.9 ± 17.3 65.0 ± 6.0
p = 0.09 70.6 ± 6.8
p = 0.14 60.4 ± 8.3
p = 0.06 87.9 ± 14.5 Triglycerides Serum 129.7 ± 17.9 116.3 ± 10.5 95.4 ± 5.7
p = 0.12 124.4 ± 8.9 111.6 ± 17.2 HDL 46.0 ± 4.6 45.8 ± 2.3 42.2 ± 2.1 45.2 ± 3.2 44.5 ± 2.5 LDL + VLDL 76.0 ± 14.7 70.5 ± 9.0 53.2 ± 4.6 79.2 ± 7.7 73.9 ± 15.4

Table 20
The content of cholesterol and triglycerides (mg / 100 ml) in rat serum on the 10th day of the experiment Indicators Groups "Cholesterol"
(n = 12) "Compound IV-1Na"
(n = 12) "Connection V-1Na"
(n = 12) "Compound III-1Na"
(n = 12) "Compound II-1Na"
(n = 11) Total cholesterol serum 144.6 ± 12.8 122.1 ± 9.8
p = 0.18 123.3 ± 7.3
p = 0.18 102.3 ± 6.9
p = 0.014 134.4 ± 12.5 HDL 46.7 ± 2.9 53.4 ± 2.2
p = 0.09 52.9 ± 3.8 48.8 ± 2.4 54.7 ± 2.2
p = 0.046 LDL + VLDL 97.9 ± 14.0 68.7 ± 10.1
p = 0.11 70.5 ± 8.9
p = 0.12 53.5 ± 7.3
p = 0.02 79.7 ± 12.6 Serum Triglycerides 115.8 ± 18.9 109.3 ± 5.1 81.9 ± 4.4
p = 0.11 109.3 ± 10.5 108.5 ± 11.3 HDL 35.5 ± 3.1 36.2 ± 2.6 31.5 ± 1.8 34.3 ± 2.3 38.9 ± 2.4 LDL + VLDL 80.3 ± 16.2 73.1 ± 5.2 50.4 ± 4.8
p = 0.1 75.0 ± 8.5 69.6 ± 11.1

The administration of mono-sodium salt of compound III to animals significantly reduced total serum cholesterol (cholesterol) by 29% and cholesterol of VLDL + LDL fractions by 40%, but did not change the level of cholesterol of antiatherogenic HDL and total serum triglycerides.

The results obtained indicate that mono- and di-sodium salts exceeded compound IV in the dynamics of action on total serum cholesterol and LDL + VLDL cholesterol and other indicators of lipid metabolism. While by the 10th day of the experiment, a reliable and comparable decrease in the aforementioned indicators occurred under the influence of all the above compounds and the Zocor comparison drug (simvastatin), the salts of compound IV began to act at an earlier stage of the experiment (on days 5 and 8) . Both sodium salts of compound IV increased HDL by the 5th day of the experiment, while compound IV increased this indicator only by the 8th day. Comparison drug simvastatin did not affect HDL cholesterol. In addition, the disodium salt of compound IV decreased the level of total serum triglycerides on days 5 and 10 of the experiment.

A distinctive property of the monosodium salt of compound V was the ability to lower the level of total serum triglycerides, while the decrease in the level of total cholesterol, VLDL cholesterol and the increase in HDL content was lower than that of the monosodium salt of compound III and the monosodium salt of compound IV.

Thus, the salts of compounds II, III, IV and V have increased lipid-lowering activity, compared with the activity of the compounds described in the publication of international application WO 99/01103, and compounds proposed in the present invention, including the ability to reduce the level of triglycerides, serum cholesterol, including in LDL and raising HDL cholesterol.

Example 19

The study of the lipid-lowering effect of compounds of the general formula (I) on the model of “endogenous” hypercholesterolemia of guinea pigs

The study was conducted on male guinea pigs (Agouti breed), with an initial weight of 304 ± 25 g. The duration of the experiment was 31 days. Control group, 6 pigs - intact animals. The studied compounds were administered from the first day of the experiment (from the first day of the introduction of the fat load).

Animals of the experimental groups received per os test compound and fat load per day for 31 days. The test compound in the following doses was administered as an aqueous solution (0.5 ml per animal); fat load - a mixture of pork fat / pre-warmed corn oil, 4: 1 by volume, at the rate of 5 ml / kg of weight 0.5 hours after administration of the test substance.

Experimental groups:

1) "control" - intact animals,

2) "fat" - animals that received only a fat load,

3) "compound IV" - animals that received a fat load + compound IV at a dose of 500 μg / kg body weight,

4) "compound V" - animals that received a fat load + compound V at a dose of 500 mcg / kg.

Data on the content of cholesterol and triglycerides in the blood serum of guinea pigs treated with a fat load and the studied compounds are presented in tables 22-25.

Table 21
Total serum cholesterol in guinea pigs treated with fat and test compounds Total serum cholesterol 28 days 31 days The control 37.4 ± 3.3 Fat 71.7 ± 14.5
n = 9 74.4 ± 11.4 Compound IV 55.5 ± 6.3
n = 10 49.2 ± 3.9
P = 0.064 Compound V 52.9 ± 6.5
n = 9 46.3 ± 4.1
P = 0.043

Statistical processing was carried out using one-way analysis of variance.

Table 22
The content of total triglycerides in the blood serum of guinea pigs treated with fat load and test compounds Total serum triglycerides 28 days 31 days The control 60.1 ± 2.4 Fat 89.7 ± 14.0 123.1 ± 35.6 Compound IV 66.8 ± 6.7 69.0 ± 13.4 Compound V 62.3 ± 5.4 56.0 ± 6.1

Table 23
The content of total cholesterol on the 31st day in the fractions of serum lipoproteins of guinea pigs receiving a fat load and test compounds Cholesterol, mmol / 100 ml General VLDL LDL The control 38.4 ± 3.3 1.6 ± 0.07 32.2 ± 2.8 Fat 74.4 ± 11.4 4.1 ± 1.2 67.6 ± 10.2
P = 0.009 Compound IV 49.2 ± 3.9 3.0 ± 0.71 42.1 ± 3.5
P = 0.040 Compound V 46.3 ± 4.1 2.1 ± 0.4 41.9 ± 2.7
P = 0.038

Table 24
The content of total triglycerides on the 31st day in the fractions of serum lipoproteins of guinea pigs treated with a fat load and the studied compounds Triglycerides, mcg / 100 ml General VLDL LDL The control 60.1 ± 2.36 38.16 ± 2.86 16.13 ± 0.97 Fat 123.2 ± 35.6 59.5 ± 19.7 42.7 ± 8.62
P = 0.015 Compound IV 69.0 ± 13.4 33.2 ± 12.3 29.6 ± 2.4 Compound V 56.0 ± 6.1
P = 0.1 22.5 ± 4.4
P = 0.1 23.1 ± 1.9
P = 0.053

Test compounds IV and V significantly reduced total cholesterol by only 31 days of the experiment by 33.9 and 37.8%, respectively. Moreover, they significantly reduced LDL cholesterol by 37.7 and 38%.

Figure 1. Change in total serum cholesterol under the influence of fat load and various doses (50-1500 μg / kg) of compound IV. Vertical thin lines indicate the standard deviation from the mean.

The advantage of the claimed compounds, in particular compounds IV, is a wide range of effective doses, which ensures the breadth of its therapeutic effect. So, for example, compound IV almost equally effectively reduced the content of total cholesterol for 20 days in the range of doses from 50 to 1500 μg / kg, differing 30 times.

Thus, the claimed compounds corresponding to the general formula (I) have significant lipid-lowering activity, significantly improving lipid metabolism in serum and in the liver.

Example 20

Comparative study of the lipid-lowering effect of compound IV and glutaryl histamine (GG, compound III WO99 / 01103) in an experimental model of Wistar rat hypercholesterolemia

Materials and research methods

The data presented are obtained after combining the results of 3 experiments .

The study was performed on 118 Wistar male rats weighing 180-230 g. Hyperlipidemia was caused by oral administration (using a gastric tube) of a cholesterol load - an oily suspension of cholesterol:

olive oil (refined, preheated for 1 h 80 ^о С) - 5 ml / kg of weight,

cholesterol (Sigma, USA) - 1g / kg of weight,

sodium cholate (Sigma, USA) - 120 mg / kg of body weight.

A cholesterol suspension was administered in the morning daily for 14 days. Animals received the test compounds (at a dose of 500 μg / kg body weight) from the first day of the experiment in the morning. All preparations were administered to animals 30 minutes before the cholesterol load.

Experimental groups:

"Cholesterol" - rats, received within 14 days per os oil suspension of cholesterol (n = 37)

1. "IV" - rats, received per os an oil suspension of cholesterol and Na salt of the test compound IV (n = 37),

2. "GG" - rats, received per os an oil suspension of cholesterol and the test compound GG / (n = 22).

Blood samples (from the tail) were taken for analysis on the 5th, 8th and 10th days of the experiment. On day 14, blood samples were obtained after decapitation. Before blood sampling, the animals starved for 10 hours.

Serum levels of total cholesterol, triglycerides, total cholesterol of high density lipoproteins (HDL) were measured. Low and very low density lipoprotein cholesterol was calculated by the difference between total serum cholesterol and HDL cholesterol.

The content of total cholesterol and triglycerides in blood serum was determined by enzymatic methods using the test kits CHOL L 250 S (No. 10003268) and TG L 250 S (No. 10003267) manufactured by Lachema (Czech Republic). The optical density was measured on a Shimadzu spectrophotometer at 500 nm, and calibration was obtained using the standard samples attached to the test kits.

Determination of the cholesterol content in high-density lipoproteins (α-LP) was carried out using the test kits “HDL CHOL 250E” (10003202) and “CHOL L 250 S” (No. 10003267) of the company “Lachema” (Czech Republic) by the method of deposition of LDL and VLDL phosphor tungsten acid and magnesium ions.

The data in tables 25-27 are presented as mean values ± standard error of the mean. The significance of differences between the “cholesterol” and “drug ...” groups was evaluated using the two-sample student t-test. The probability of error (p) is indicated in the columns of the tables.

Research results

Experimental Hypercholesterolemia

The initial level of total cholesterol and plasma triglycerides is presented in Table 25. The dynamics of lipid metabolism changes are in Table 26.

Table 25
Rat lipid metabolism (n = 24) before experiment Initial biochemical parameters, mg / 100 ml (n = 30) Total cholesterol Serum 72.3 ± 1.8 HDL 36.1 ± 1.7 LDL + VLDL 36.2 ± 2.2 Cholesterol Atherogenic Index 1,07 ± 0,05 Triglycerides Serum 69.0 ± 5.7 HDL 15.4 ± 1.3 LDL + VLDL 53.6 ± 4.9 Triglyceride index 3.58 ± 0.22

The influence of the studied compounds on the development of hypercholesterolemia

Data on the effect of the studied compounds on the cholesterol and triglycerides in the blood serum of rats receiving cholesterol load are presented in tables 26 and 27.

Table 26
Changing the parameters of lipid metabolism throughout the experiment HDL total cholesterol initially 5 days 8 days 10 days 14 days "Cholesterol" 36.1 ± 1.7 31.7 ± 1.6 27.3 ± 1.4 35.7 ± 1.5 29.9 ± 1.9 GG 26.4 ± 1.7
P _cold-yy = 0.03 23.5 ± 1.0
P _cold-yy = 0.06 32.2 ± 1.0
P _cold-yy = 0.04 25.4 ± 1.4
P _cold-yy = 0.06 IV 33.3 ± 1.6
P _gg-iv = 0.005 30.1 ± 1.4
P _{Hall IV} = 0.07
R _yy-iv = 0,0003 41.6 ± 2.1
P _chol-IV = 0.03
P _gg-iv = 0.0001 30.1 ± 1.6
P _gg-iv = 0.04 LDL total cholesterol "Cholesterol" 36.2 ± 2.2 79.3 ± 4.7 76.3 ± 4.1 81.2 ± 6.9 100.9 * ± 9.5 GG 64.9 ± 3.1
P _cold-gg = 0.01 70.5 ± 2.9 61.0 ± 3.6
P _cold-gg = 0.01 65.3 ± 6.5
P _cold-gg = 0.003 IV 69.2 ± 3.3
P _{Hall IV} = 0.08 66.9 ± 4.3
P _{Hall IV} = 0.12 65.8 ± 4.1
P _{Hall IV} = 0.06 69.2 ± 5.3
P _chol-IV = 0.005 Cholesterol Atherogenic Index (total LDL + VLDL / HDL cholesterol) "Cholesterol" 1,07 ± 0,05 2.9 ± 0.3 3.3 ± 0.3 2.7 ± 0.4 4.2 ± 0.7 GG 2.8 ± 0.3 3.1 ± 0.1 1.9 ± 0.1
P _cold-yy = 0.06 2.7 ± 0.3
P _cold-yy = 0.04 IV 2.3 ± 0.2
P _{Hall IV} = 0.1 2.6 ± 0.3
P _{Hall IV} = 0.07
P _gg-iv = 0.02 1.6 ± 0.1
P _chol-IV = 0.008
P _gg-iv = 0.08 2.6 ± 0.3
P _chol-IV = 0.03

Table 27
Significant (p ≤ 0.05) differences in changes in the parameters of lipid metabolism between groups IV and GG according to the t-test t-test Differences in changes in lipid metabolism parameters in groups days GG (n = 22) IV HDL cholesterol 5 + 22% 8 + 24% 10 + 26% fourteen + 15.7% Cholesterol Atherogenic Index (total LDL + VLDL / HDL cholesterol) 5 8 - fifteen% 10 - eleven% Designations: - 10% - significantly lower by 10%. For 100%, the level in the Cholesterol group is accepted.

The data presented in tables 25-27 clearly indicate that compound IV increases the level of anti-atherogenic HDL cholesterol by 10-14% compared with the control, while a significant decrease in HDL cholesterol occurred under the influence of glutaryl histamine.

As a result, the decrease in the cholesterol atherogenicity index by glutaryl histamine was due to a decrease in the cholesterol of all plasma fractions, and HDL and LDL + VLDL.

Compound IV compares favorably with glutaryl histamine by a significant significant decrease in the cholesterol atherogenic index by 20-40% during the experiment, precisely due to an increase in the HDL fraction.

Comparison of the hypolipidemic effect of glutaryl histamine with the corresponding activity IV showed that compound IV according to the invention significantly exceeded glutaryl histamine in increasing HDL cholesterol and lowering atherogenicity index (Table 27).

Example 21

Studies of the anti-inflammatory activity of compounds of the general formula I (1% gel) in vivo model of carrageenan edema

Materials and methods

Tests are performed on non-linear white mice weighing 30-32 g. A model of carrageenin-induced edema is used according to the method described in Winter et al. In: De Rosa M., Giroud J.P., Willoughby D.A. Studies of the mediators of the acute inflammatory response induced in rats in different sites by carrageenan and turpentine. // J. Phamacol., 1971, V.104, p. 15-29. 1% carrageenin solution (SERVA) in a volume of 0.05 ml is subplanted into the right paw of the mouse. Substances in the form of a 1% gel are applied to the paw 2 times: the first time - immediately after the administration of carrageenin; the second time - after 1.5 hours. The measurement of the volume of the right and left (intact) paws is carried out using a caliper 3 hours after the introduction of carrageenin. The inflammatory response and the effect of therapeutic effects are evaluated by the formula

Where

P - increase in edema,

O is the volume of the paw after the introduction of phlogogen,

And - the volume of the paw before the introduction of phlogogen.

The effect of therapeutic effect is evaluated by the degree of suppression of the inflammatory reaction compared to the control and calculated by the formula

Where

(o) - treated animals,

(k) - untreated animals.

Table 28
The effect of compounds of the general formula I (1% gel) on the development of carrageenin edema of mouse paws Test substances Paw Volume Difference Volume Growth (%) Inhibition of inflammatory response (%) V 0.104 ± 0.009 ** 60.0 43.8 V-1Na 0.14 ± 0.02 ** 79.0 26.2 IV-1Na 0.105 ± 0.002 ** 47.6 33.0 II-1Na 0.1 ± 0.009 ** 32,3 51.6 III-1Na 0.15 ± 0.01 * 50,0 25.0 Voltaren 0.08 ± 0.006 ** 25.8 61.3 * - statistically significant relative to the model * - P <0.01
** - P <0.01

The experimental results presented in table 28 demonstrate the pronounced anti-inflammatory activity of the monosodium salt of compound II, comparable with the activity of the reference drug - voltaren.

Compound V and its monosodium salt exhibit moderate activity in this model of carrageenin edema of mouse paws, inhibition of edema is 43.8% and 26.2%, respectively.

Thus, the whole complex of experimental models used indicates a significant anti-allergic, anti-anaphylactic and anti-inflammatory activity of compounds of the general formula I, which is manifested both in in vitro tests and in modeling of allergic and inflammatory pathology in vivo. It is shown that the described compounds corresponding to the general formula I exhibit a significant protective effect with respect to the systemic anaphylactic reaction in vivo under the conditions of modeling allergic rhinitis, in addition, these compounds suppress eosinophilic inflammation and have a distinct anti-inflammatory effect.

Examples of dosage forms

Example 22

A. Tablet form

A tablet form is prepared using the following ingredients:

A compound corresponding to general formula (I) or a pharmaceutically acceptable salt thereof 1-100 mg Potato starch 20-50 mg Magnesium stearate 3 mg Aerosil 1 mg Lactose up to 300 mg

The components are mixed and compressed to form tablets weighing 300 mg each.

B. Suppositories

Suppository composition example:

A compound corresponding to general formula (I) or a pharmaceutically acceptable salt thereof 1-100 mg Cacao butter amount needed to get a suppository

If necessary, it is possible to produce rectal, vaginal and urethral suppositories with appropriate excipients.

V. Ointment

An example of the composition of the ointment:

A compound corresponding to general formula (I) or a pharmaceutically acceptable salt thereof 0.01-0.1 g Petroleum jelly 10 g

Ointments are made according to well-known technology.

G. Gels

Gel composition example:

A compound corresponding to general formula (I) or a pharmaceutically acceptable salt thereof 1-100 mg Carbopol 200 mg Benzyl alcohol 20 mg Ethanol 300 mg Water up to 10 g

D. Dry powder for inhalation

Powder Composition Example:

A compound corresponding to general formula (I) or a pharmaceutically acceptable salt thereof 0.2 g Lactose up to 1 g

The powder is placed in a special device (container) or in a gelatin capsule.

E. Nasal Spray

An example of the composition of the spray:

A compound corresponding to general formula (I) or a pharmaceutically acceptable salt thereof 1.5-150 mg Purified water up to 15 ml

E. Eye drops

Drop composition example:

A compound corresponding to general formula (I) or a pharmaceutically acceptable salt thereof 0.5-50 mg Preservative 10 mg Purified water up to 5 ml

E. Injection

An example of the composition of the solution for injection:

A compound corresponding to general formula (I) or a pharmaceutically acceptable salt thereof 0.2-20 mg Water for injections 2 ml

Claims (2)

1. The method of obtaining N-acyl derivatives of amino acids of General formula (I)

where n is 2 or 3; and
R represents

or their pharmaceutically acceptable salts, including the addition of glutaric or succinic anhydride as a solid to an aqueous solution of an amino acid of the general formula

where R represents

and, optionally, converting the desired product into its salt. 2. The method of obtaining N-acyl derivatives of amino acids of General formula I

where n is 2 or 3; and
R represents

or their salts, including carrying out the reaction in a two-phase system of glutaric or succinic anhydride in a water-immiscible organic solvent with an aqueous solution of an amino acid of the general formula

or its salt,
where R represents

and, optionally, converting the desired product into its salt.