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

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

 

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

Prior art

As you know, currently allergic diseases and disorders of lipid metabolism are very common due to poor environmental conditions, changing eating patterns and lifestyle of the population. Therefore, the problem of drugs to combat these diseases, as well as with inflammatory processes, generally accompanying allergies, continues to be relevant.

In this regard, of particular interest are compounds comprising residues of substances of natural origin, as they can be predicted lower toxicity and frequency of side effects.

In the published international application WO 99/01103 described antiallergic and hypolipidemic action of N-acyl derivatives of biogenic amines, for example, γ-glutamylcysteine, and its nearest analogue of glutamylcysteine that are most similar in structure and action of the claimed compounds. In article Krzeczkowska CENTURIES, Zheltukhina GA, Nebolsin V.E. and others Studied the e antianaphylactic activity and mechanisms of action of γ-L-glutamylcysteine. // Pathogenesis, 2003, Vol. 1, No. 2, p. 60-64 shown that γ-glutamylcysteine has expressed antianaphylactic activity using different animal species and routes of administration. The results obtained indicate that in the fat cells of animals under the action of γ-glutamylcysteine significantly reduced the content of histamine and antigen-stimulated secretion. In the test on investigating the influence of glutamylcysteine on the severity of antigen-induced bronchoconstriction has been shown to reduce the magnitude of bronchoconstriction by more than 50% compared with control. This effect was manifested as oral and intratracheal method of its introduction in a low dose of 50 mcg/kg. Glutamylcysteine has the ability to reduce the symptoms of passive cutaneous anaphylaxis from 34 to 42% and thus exceeds the efficiency of suprastin, but inferior to claritin. In WO 99/01103 shown that the introduction of animals glutamylcysteine at doses of 50 and 500 mg/kg demonstrated a significant decrease in the intensity of delayed-type hypersensitivity. In addition, glutamylcysteine at doses of 50 and 500 mg/kg also had some anticholesterolemic activity, reducing the total cholesterol compared to animals with atherogenic load of 5-7%.

Lack of glutamylcysteine is it with anicello high cost and low availability of feedstock to obtain - histamine. In addition, the substance is not effective in these tests.

With the aim of expanding Arsenal of technical tools and create a more effective and affordable anti-allergic, anti-inflammatory and lipid-lowering means, the inventors have identified some specific N-acyl derivatives of amino acids of General formula (I)disclosed in published international application WO 99/01103, but specifically it is not described, is not received and is not described.

So, the General formula (I) above international application fall compounds of the present invention Nα-succinyl-L-tryptophan (II), Nα-glutaryl-L-tryptophan (III), Nα-glutaryl-L-histidine (IV) and Nα-succinyl-L-histidine (V). However, in this publication is not given any specific structural formulas of these compounds, nor any physico-chemical characteristics, and also does not describe the methods for their preparation. In the published international application WO 03/072124 formulae Nα-glutaryl-L-tryptophan (II), Nα-glutaryl-L-histidine (IV) and Nα-succinyl-L-histidine (V), but not described method of their synthesis and not given their physico-chemical constants.

One of the connections glutamylcysteine is mentioned only in the form of a methyl ester at the C-end of His [Glt-His(OMe)] in U.S. patent 3963691, ka is este intermediate compounds in the synthesis of peptide Glt-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-poly-Lys.

Connection succinylated mentioned in the published international application WO 93/04690. In this publication indicated that adding free imidazole or succinyldicholine accelerates the interaction of carnosine with dihydroxyacetone. Methods of synthesis of succinyldicholine and its physico-chemical constants are not shown.

The structural formula of succinyldicholine mentioned in the application form USA2005079515. In this publication, neither the methods of synthesis of succinyldicholine nor its physico-chemical constants are not shown.

Obtaining Nα-glutaryl-L-histamine described in published international application WO 99/01103 and represents the N-acylation of biogenic amine glutaric anhydride in the medium of anhydrous N,N-dimethylformamide. In addition, the publication herskowitz A.A., Kibirev VK // Chemical synthesis of peptides. / Kyiv: Naukova Dumka, 1992, 360, describes how the acylation of amino acids in aqueous-organic, strongly alkaline environment.

In F. Sorm, Pravda Z., Proteins and amino acids. X. Synthesis of two peptide analogs. // Chemicke Listy pro Vedu a Prumysl., 1951, V.45, p.423-425 described method of synthesis of succinylcholine ethyl ester in a mixture of water and ethyl acetate at a ratio of (1:1) on the basis of the hydrochloride of the ethyl ester of tyrosine and glutaric anhydride in the presence of NaHCO3to maintain a slightly alkaline pH.

However, the acylation with anhydrides of dicarboxylic acids free Geest is Dina are not described in literature.

The aim of the present invention are new ways of efficient synthesis of N-acyl derivatives of amino acids having anti-allergic, anti-inflammatory and hypolipidemic effect in low doses and does not exhibit side effects.

The inventors have developed a simple preparative method of synthesis of compounds of General formula (I), namely, that the glutaric anhydride or succinic acid in the form of a solid substance is added to aqueous solution of amino acids in the absence of inorganic and organic bases to obtain the target product with a sufficiently high yield of 55-60%.

The inventors have also developed another method for the synthesis of compounds of General formula (I), including N-acyl derivative of histidine, comprising conducting the reaction in a biphasic system consisting of an aqueous solution of histidine and solution of the anhydride in a suitable organic solvent using an excess Alliluyeva agent.

Brief description of the invention

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

where n is 2 or 3; and

R represents

and their salts, including the addition of glutaric anhydride or succinic sour the s in the form of solids to aqueous solution of amino acids of General formula:

or its salts, where

R represents

not necessarily, the transformation of the target product in its salt.

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

where n is 2 or 3; and

R represents

and their salts, comprising conducting the reaction in a biphasic system with glutaric anhydride or succinic acid in a water-immiscible organic solvent with an aqueous solution of the amino acid of General formula:

or its salts, where

R represents

and, optionally, the transformation of the target product in its salt.

A detailed description of the invention

Preferred compounds of General formula I are presented in table 1.

Table 1
ConnectionNo. of connectionsRn
Nα-succinyl-L-tryptophanII(Ind) 2
Nα-glutaryl-L-tryptophanIIIInd3
Nα-glutaryl-L-histidineIV(Im)3
Nα-succinyl-L-histidineVIm2

The synthesis of compounds of General formula I can be carried out in two ways. The first way is to gradually added to the aqueous solution of amino acids of General formula

or its salts, where

R represents

glutaric or succinic anhydride in the form of a solid substance, followed by separation of the target product ion-exchange chromatography, preferably by passing the reaction mixture through a column of cation exchange resin and subsequent crystallization from aqueous solution. The obtained crystals of the target product is washed with a suitable solvent, preferably methanol. The main advantage of the proposed method consists in the absence of alkali in the aqueous solution of amino acids, which prevents inactivation of the anhydride of dicarboxylic acid is the result of hydrolysis. In addition, the balance of imidazole in the composition of amino acid molecules may carry out acid-base autocatalyst reaction of acylation of the amino group of amino acids. Relatively high yields (55-60%) when using the proposed method is achieved, in particular, due to gradual addition of an anhydride of dicarboxylic acids taken in excess, and intensive mixing of the reaction mass.

Compounds of General formula I can also be obtained in an alternative way in the two-phase system, including the addition of glutaric anhydride or succinic acid in a water-immiscible organic solvent to aqueous solution of amino acids of General formula:

or its salts, where

R represents

This method allows you to use excess Alliluyeva agent, to achieve a complete acylation of the α-amino group of the amino acid and the yield of the target product is about 70%. To maintain the required pH instead of inorganic alkalis are used, an organic base is pyridine, which is not hydrolyzes anhydride, and, in addition, as is known, is a catalyst for acylation. The use of pyridine avoids contamination of the final product inorganic salts, which, together with the reaction product remains in the aqueous layer. Use the bathrooms approaches can simplify the separation of the target product from the unreacted anhydride and the appropriate amino acids and select the target product by simple crystallization.

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

Preferred solvents used for crystallization of the desired product are water-alcohol mixture, in particular water-ethanol.

Compounds of General formula I can also be obtained in the form of pharmaceutically acceptable salts with sodium hydroxide, potassium hydroxide, magnesium carbonate, lithium hydroxide, calcium carbonate routine methods widely described in the literature.

Compounds of General formula I possess anti-allergic, anti-inflammatory and hypolipidemic activity and can be used for treatment of allergic, anaphylactic, including inflamed diseases and disorders of lipid metabolism.

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

Compounds of the present invention are introduced in an effective amount that provides the desired therapeutic result.

For the treatment of allergic diseases, including bronchial asthma, allergic rhinitis, allergic rhinitis, seasonal rhinitis, perennial rhinitis, atopic dermatitis, psoriasis, urticaria, allergic (including anaphylactic) reactions to insect stings and medications, cold Allergy, allergic conjunctivitis, chronic obstructive lung diseases, namely chronic obstructive bronchitis, emphysema, obliterative bronchitis, cystic fibrosis and diseases associated with lipid metabolism disorders, such as atherosclerosis, obesity, coronary heart disease and cerebral, myocardial infarction, stroke, compounds of General formula I can be introduced oral, topical, parenteral, intranasal, inhalation and rectal in the form of standard dosage forms containing non-toxic pharmaceutically acceptable carriers. Used in the present description, the term "parenteral administration" means in the skin, intravenous, intramuscular or intrathoracic injection or infusion.

Compounds of the present invention can be administered to the patient in doses, comprising from 0.01 to 10 mg/kg of body weight per day, preferably at doses from 0.05 to 5 mg/kg once or more than once a day.

It should be noted that the specific dose for each particular patient will depend on many factors, including the activity of this used compound, the age, body weight, sex, General health and diet of the patient, time and route of administration of drugs, the rate of its excretion from the body, specifically used a combination of drugs and the severity of the disease in the individual being treated.

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

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

The identity of the compounds was checked by TLC on plates "Kieselgel 60 F254" Merck (Germany) systems: methanol (1), chloroform-methanol-ammonia(4:3:1) (2).

The chromatogram showed chlorthalidone reagent, ninhydrin, th is house to glow in UV light.

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

1H-NMR were recorded on a device "AMX-400 Bruker (Germany).

The melting point was determined on the device "Boetius (Germany).

Analytical HPLC sang and danced on the device "System Gold" ("Beckman, USA): the rate of elution of 0.25 ml/min, detection at 214 nm in the conditions: column Ultrasphere ODS "Beckman", 2×250 mm, 5 μm, elution with 0.1%TFA, the rate of elution of 0.25 ml/min (1); the rate of elution 1 ml/min, detection at 220 nm, column Luna 5 "Phenomenex, C18, 250×4.6 mm, elution with 25% acetonitrile in 0.05 M phosphate buffer (pH 3.0) (2).

Example 1

Nα-Glutaryl-L-histidine (IV)

Method And

To a solution of 103,4 g (0.67 mol) of histidine in 400 ml of water is added to 83.7 g (0.73 mol) of glutaric anhydride. The suspension is stirred for 1 hour, the resulting solution is evaporated to a volume of 150 ml, stored in the refrigerator for 16 hours. The precipitation is filtered off, washed with 150 ml of methanol and dried. Cleaning is performed by ion-exchange chromatography on resin Purolite N+form elwira water. The fractions containing the desired product are pooled, evaporated prior to the deposition of sediment and left for 16 hours at +4°C. the precipitation is filtered off, washed with 200 ml of methanol and dried to constant weight. Output 98,8 g (55%). Rf0,55 (1), 0,37 (2). TPL= 222-224°C. [α]D20+15, 95° (C 0,53, water). [M+H]+270,1 1H-NMR spectrum (D2O), δ, ppm: 1,60-1,80 (m, 2H, β-CH2-Glt), 2,10-of 2.25 (m, 4H, α,γ-CH2-Glt), 2,90-of 3.25 (m, 2H, β-CH2-His), 4,40-4,50 (m, 1H, α-CH-His), to 7.15 (s, 1H, CH-4-Im), and 8.50 (s, 1H, CH-2-Im). Found, %: C 49,18; H 5,91; N 15,42. C11H15N3O5. Calculated, %: C 49,07; H 5,62; N 15,61.

Method B

To a suspension of 0.3 g (of 1.93 mmol) of histidine in 5 ml of water with vigorous stirring of 0.44 g (3,86 mmol) of glutaric anhydride, dissolved in 2.5 ml of ethyl acetate. Stirred for 2 hours, the pyridine was adjusted pH to 7 and stirred for further 1 hour. An ethyl acetate and the aqueous layer was separated. The aqueous layer was washed twice with ether, the ether layer discarded. Water is removed in vacuum, the residue is dissolved in a minimum amount of water and add ethanol until the beginning of precipitation of a white precipitate, leave at 4°C for 20 hours the Precipitate was separated by filtration, dried in vacuum. Yield 0.36 g (70%). Rf0,56 (1), 0,35 (2). TPL= 219-221°C. [α]D20= +15,71° (C 0,56, water). [M+H]+270,1.1H-NMR spectrum (D2O), δ, ppm: 1,40-of 1.55 (m, 2H, β-CH2-Glt), 1,90-2,0 (m, 4H, α, γ-CH2-Glt), of 2.7-3.0 (m, 2H, β-CH2-His), 4,20-4,30 (m, 1H, α-CH-His), 6,95 (c, 1H, 4-CH-Im), 8,30 (c, 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. C11H15N3O5. Calculated, %: C 49,07; H 5,62; N 15,61.

Example 2

Nα-Succinyl-L-histidine (V)

The synthesis was carried out according the method And, provided for connection of the IV.

Yield 0.08 g (57%).

Rf0,44 (1), 0,25 (2).

TPL= 179-181°C.

[α]D20= +30,71° (C 0,56, water).

[M]+255,2.

1H-NMR spectrum (D2O), δ, ppm: 2,15-of 2.30 (m, 4H, (CH2)2-Suc), of 2,75 2,95 (m, 2H, β-CH2-His), 4,25 (USS, 1H, α-CH-His), 6,95 (c, 1H, 4-CH-Jm), 8,25 (c, 1H, 2-CH-Jm).

Found %: C 47,09; H 5,04; N 16,40. C10H13N3O5. Calculated %: C 47,06; H 5,13; N 16,46.

The synthesis was carried out according to method B described for compound IV.

Output 0,101 g (67%).

Rf0,45 (1), 0,27 (2).

TPL= 178-180°C.

[α]D20= +30,8° (C 0,57, water).

HPLC under the conditions (1) - individual peak, retention time 7,54 minutes

Found %: C 47,15; H 5,2; N 16,50. C10H13N3O5. Calculated %: C 47,06; H 5,13; N 16,46.

Example 3

Nα-Glutamylation (III)

To a suspension of 1.0 g (4.9 mmol) of tryptophan in 7 ml of water are added dropwise a solution of 1 N NaOH (4.9 mmol). To the resulting solution was added a solution of 0.56 g (4.9 mmol) glutaric anhydride in 3 ml of ethyl acetate. The reaction mixture is stirred for 3 hours at room temperature in argon atmosphere in the dark, left for 16 hours at +4°. The solvent of the reaction mixture is removed under vacuum. The obtained oily residue is dissolved in 30 ml of water under stirring, cooled to 0°C, add a solution of 1 N HCl to pH 4. The product was EXT Airout with ethyl acetate (3×25 ml). United an ethyl acetate extract is cooled to 0°, washed with water (4×25 ml) to pH 7, the solution of 5% HCl (5 ml), water (4×25 ml) to pH 7, dried over anhydrous Na2SO4within 1 hour. Sediment Na2SO4filtered off, washed with ethyl acetate, the solvent is removed in vacuum. Get a grayish solid residue, which is dried in vacuum.

Yield 1.0 g (70%).

Rf0,54 (1).

TPL= 150-152°C.

[α]D20= +8,20° (C 0.5, methanol).

1H-NMR spectrum (CD3OD), δ, ppm: 1,75-of 1.84 (m, 2H, β-CH2-Glt), 2,15-of 2.30 (m, 4H, α,γ-CH2-Glt), 3,30 is 3.40 (m, 2H, β-CH2-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)to 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 minutes

Found, %: 60,07; H 5,65; N Is 8.75. C16H18N2O5Calculated, %: C 60,37; H 5,7; N 8,8.

Example 4

Nα-Succinyl-L-tryptophan (II)

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

The output of 100.5 mg (67%).

Rf0,63 (1).

[α]D20= +21,05° (C 0,6, water).

1H-NMR spectrum (DMSO-d6), δ, ppm: 2,33-to 2.41 (m, 4H, α,β-CH2-Suc), 2,93-a 3.01 (m, 1H, β-CH2-Trp), 3,10 -, and 3.16 (m, 1H, β-CH2-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) - indiv the dual peak, retention time 6,35 minutes

Found, %: C 59,07; H 5,65; N 9,35. C15H16N2O5Calculated, %: C 59,21; H 5,3; N Of 9.21.

Example 5

Monosodium salt Nα-glutaryl-L-histidine (IV)

To a solution of 1.0 g (3.7 mmol) of Nα-glutaryl-L-histidine in 15 ml of water under stirring and cooling to +5°C. add a solution of 0.15 g (3.7 mmol) of NaOH in 20 ml of water. The solution is stirred for 30 min, the solvent is removed in vacuum. To the oily residue are added in several portions of benzene, the solvent is removed in vacuum. The solid residue is dried over granular alkali.

Yield 1.07 g (99.7 per cent).

TPL= 208-210°C.

[α]D20= +16,27° C 0,58, water).

Found, %: C 45,25; H 5,51; N 14,52. C11H15N3O5Na. Calculated, %: C 45,21; H 5,17; N 14,38.

Example 6

Monosodium salt Nα-succinyl-L-histidine (V)

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

Yield 1.06 g (97,0%).

[α]D20= +40,21° (C 0,48, water).

Found, %: C 43,25; H 4,51; N 15,52. C10H13N3O5Na. Calculated, %: C 43,17; H 4,71; N 15,10.

Example 7

Monosodium salt Nα-succinyl-L-tryptophan (II)

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

Ihad 0.21 g (98,0%).

TPL= 147-150°C.

[α]D20= +22,02° (C 0,39, water).

Found, %: C 55,25; H 4,51; N 8,32. C15H16N2O5Na. Calculated, %: C 55,05; H Is 4.93; N 8,56.

HPLC under the conditions: (2) individual peak, retention time 6,56 minutes

Example 8

Monosodium salt Nα-glutaryl-L-tryptophan (III)

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

Yield 0.11 g (99,0%).

TPL= 128-130°C.

[α]D20= +representing 22.06° (C 0,34, methanol).

Found, %: C 56,15; H To 5.21; N By 8.22. C16H18N2O5Na. Calculated, %: C 56,30; H 5,32; N 8,21.

HPLC under the conditions: (2) individual peak, retention time of 6.96 minutes

Example 9

The disodium salt of Nα-glutaryl-L-histidine (IV)

To a solution of 1.0 g (3.7 mmol) of Nα-glutaryl-L-histidine in 15 ml of water under stirring and cooling to +5°C. add a solution of 0.3 g (7,44 mmol) of NaOH in 15 ml of water. The solution is stirred for 30 min, the solvent is removed in vacuum. To the oily residue are added in several portions of benzene, the solvent is removed in vacuum. The solid residue is dried over granular alkali.

The yield of 1.15 g (99,0%).

[α]D20= +11,92° (C 0,57, water).

Found, %: C 41,25; H 4,51; N 13,52. C11H15N3O5Na2. Calculated, %: C 41,91; H 4,80; N 13,3.

Example 10

Dint Ieva salt N α-succinyl-L-histidine (V)

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

The output of 1.16 g (99,0%).

TPL= 124 to 128°C.

[α]D20= +grade of 20.06° (C 0,67, water).

Found, %: C 39,55; H Or 4.31; N 13,52. C10H13N3O5Na2. Calculated, %: C 39,88; H 4,35; N 13,95.

Example 11

The disodium salt of Nα-succinyl-L-tryptophan (II)

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

Yield 0.56 g (97.7 percent).

Found, %: C 51,35; H Or 4.31; N By 8.22. C15H16N2O5Na2. Calculated, %: C 51,43; H 4,60; N 8,0.

Example 12

The disodium salt of Nα-glutaryl-L-tryptophan (III)

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

Yield 0.56 g (98.5 per cent).

Found, %: C 52,55; H 4,71; N 7,52. C16H18N2O5Na2. Calculated, %: C 52,75; H To 4.98; N 7,69.

Tests for biological activity

Example 13

The effect of compounds of General formula I at allergic reactions of immediate type (test induced by ovalbumin (OA) degranulation of basophils in the blood of immunized Guinea pigs in vitro)

The selection of leukocytes from the blood sea St. the NCI has implemented method Primes (Immunological methods./Ed. Grimes/ - M.: Medicine, 1987, str in our modification).

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

Under ether anesthesia from the heart of the Guinea pig were collected 15 ml of blood. For selection of basophils in the composition of the leukocyte suspension used double deposition of cells by EDTA and using nitratsoderzhaschie precipitating liquid.

The blood was mixed with 5% EDTA·Na22H2O (Sigma) in a ratio of 9:1 and after 30 minutes, gently centrifuged (12 min at 80 g). The supernatant was collected and centrifuged 15 min at 500 g.

The remaining blood cells were added nitratsoderzhaschie precipitating liquid (3) in a ratio of 3:10 (thermostatically at 37°C for 30 min). Enriched leukocytes supernatant fraction was centrifuged for 7 min at 100 g. To the precipitate of cells was added to a 0.85% NaCl solution, and the cell concentration was brought to 30×103/µl.

Staging test degranulation of basophils in vitro[Handbook of clinical laboratory methods./Edited Eaast/ - M.: Medicine, 1975, page 130].

For the production test in the centrifuge tube (used 3 tubes on to the introduced each sample) were placed 300 µl of cell suspension, then add 300 ál of salt solution of tested compound (or saline in the control of spontaneous and maximal degranulation) and preincubated at 37°C for 15 min, then was added 300 μl of 1% saline OA in each tube (in the control of spontaneous degranulation was added to the saline solution in the same amount) and again was preincubated at 37°C for 10 min. working concentration of leukocytes is with 104/µl. From each tube was sampled (100 μl) in a separate test tube for an assessment of the full degranulation of basophils, and the remaining cells were added to the cooled saline solution (5 ml in each tube) to stop reaction degranulation, and then were centrifuged for 7 min at 100 g, and the precipitate was preparing preparations for microscopic examination. Fixation and coloration of the drugs was performed by the method Seder et al. (R.A. Seder 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 specific grain basophils used dye 0,5% Allenby blue (pH 1.0)and kernel they finished painting areas safranin (0.1% solution in 1% acetic acid). Drugs used to estimate total inhibition of degranulation

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

where

the Mach - %degranulated basophils at maximum degranulation (OA),

spent. - % degranulated basophils in spontaneous degranulation (control),

the experts. - % degranulated basophils after exposure to the compounds.

A full assessment of degranulation of basophils

Selected after setting the test degranulation of basophils samples (100 μl) were placed in tubes with dye (0.5% of Allenby blue, pH 1.0) 1:1 ratio. The painting was made at a room temperature not less than 50 minutes counting the number of stained basophils was performed using a camera Fuchs-Rosenthal. Braking is full of degranulation (TPD) 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 (3 samples) the number of basophils in the test spontaneous degranulation;

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

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

Table 2
Inhibition of OA-induced degranulation of basophils in the blood of immunized Guinea pigs in vitro under the influence of the compounds of General formula I
no experience n/p GroupBraking is full of degranulation (TPD), (%)The number of completely degranulated basophils, (%)
1.Control 1 (spontaneous degranulation)1000
2.Ovalbumin 1%
(max degranulation)
035,2±0,8
3.Compound IV
(10-3M)
to 99.2±11,22,6±2,6*
4.Compound IV
(10-4M)
99,5±12,02,9±1,6*
5.Compound IV
(10-5M)
113,5±2,70
6.Compound IV
(10-6M)
90,0±1,83,9±0,6
7.Compound IV
(10-7M)
76,7±1,59,1±0,8
8.CH is tailgatin
(10-3M)
9,3±5,531,6±6,33
9.Glutamylcysteine
(10-4M)
24,1±1,125,3±3,38
10.Glutamylcysteine
(10-5M)
029,8±6.73 x
11.Control 21000
12.Ovalbumin 1%
(max degranulation)
038,9±8,43
13.Connection V
(10-3M)
10,6±7,635,3±7,29
14.Connection V
(10-4M)
18,9±11,831,2±6,8
15.Connection V
(10-5M)
44,0±11,2724,6±10,38
16.Hydrocortisone
(10-3M)
71,0±1,610,±0,7
17.Hydrocortisone
(10-4M)
48,0±0,817,3±0,9
18.Hydrocortisone
(10-5M)
40,0±0,620,0±1,3
(* - P < 0,001)

The data in table 2 show that, compared with glutamylcysteine compound (IV) has expressed antianaphylactic effect, manifested almost 100% inhibition of the degranulation test full OA-induced degranulation of basophils blood actively immunized Guinea pigs (reaction anaphylaxis in vitro in ascallaway environment). Significant antianaphylactic the effect of compound IV is manifested in the decrease in the number degranulating cells, especially pronounced at a concentration of 10-5M (no degranulating cells).

Example 14

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

Used the model of bronchospasm in generalizirovanny actively sensitized Guinea pigs with aerosol exposure of ovalbumin as an antigen (V.L. Kovalev "guidelines for the study of bronchodilators, mucolytic and protivovesa the tion of funds".//Manual on experimental (preclinical) study of new pharmacological substances, Moscow, 2000, S. 242-250).

Guinea pigs were senzibilizirani the ovalbumin method Andersson (P. Anderson, Antigen-induced bronchial anaphulaxis in actively sensitized geinea-pigs.//Allergy, 1980, Vol.35, p. 63-71) and 1-2 months after sensitization induced bronchospasm aerosol introduction resolution doses of ovalbumin (3 mg/kg in 1 ml of saline).

In the experimental groups of Guinea pigs within three days intragastric tube has introduced the compounds in doses of 10 ág/kg 50 ág/kg 150 ág/kg In another series of experiments the compounds at a dose of 50 mcg/kg (in 1 ml saline) was administered inhalation (via nebulizer) within three days 1 time per day. The control group was administered saline. Within 1 hour after the last injection of substances was ingalirovti using the nebulizer ovalbumin and estimated duration (in seconds) and the intensity of bronchospastic reactions of animals.

Table 3
The braking system anaphylactic reaction Guinea pigs inhalation introducing the compound IV at a dose of 50 mg/kg
GroupThe duration of the acute phase, sThe duration of the subacute phase, s
Control 1
(saline)
180±6650±34
Compound IV
50 ág/kg
0400±25

Table 4
The braking system anaphylactic reactions of Guinea pigs after intragastric administration of compound IV in doses of 10 and 150 µg/kg (M±m)
GroupThe duration of the acute phase, sThe total reaction time, sec
Control 2
(saline)
296,7±104,6628,3±80,6
Compound IV
10 ug/kg
68,0±54,7*428,0±75,0
Compound IV
150 ág/kg
72,0±42,1*337,0±78,5*
* - P < 0,001

The results of the experiments are presented in tables 3 and 4 show that compound IV after intragastric administration at doses of 10 and 150 mcg/kg and inhalation dose of 50 mg/kg showed antianaphylactic activity. Inhalation introduction of a substance at a dose of 50 mg/kg blocked the development of acute phase b is egocentricities reaction, which, causing asphyxia, is a cause of death. After intragastric administration of compound IV in doses of 10 mg/kg and 150 mg/kg was identified significant protective effect against antigen-induced bronchoconstriction.

Thus, compound IV has a significant protective effect against systemic anaphylactic reactions in vivo.

Example 15

Anti-allergic activity of the compounds of General formula I on the model of allergic rhinitis in Guinea pigs

Used model of allergic rhinitis in Guinea pigs.

Guinea pigs were immunized according to a certain scheme for 1.5-2 months (Hutson P.A., Church, M.K. et al., 1988): initially, animals were immunized by intraperitoneal injection of ovalbumin in the dose of 10 mg/kg at 7-day intervals (twice), and then the pigs were ingalirovti using a nebulizer equipment (Pari) a solution of ovalbumin in increasing concentrations, ranging from 0.1% to 1% with an interval of 4 days between inhalations. The last dose of ovalbumin was administered in the nasal passages using a micropipette. 24 hours after the last injection of ovalbumin produced the fence nasal rinse (through the system of special tubes) and changes in the nasal mucosa was assessed by using a range of methods: histological and cytological. The compounds (0.1% solution) was administered daily is by inhalation using a nebulizer equipment for 6 days, on the 6th day of injection caused the provocation antigen (OA). Nasal wash was received the next day after the provocation.

Table 5
The effect of compound IV on lymphocytosis (absolute number of cells in 1 μl) in natalem flush
GroupIntact controlModel + provocation (rhinitis)Compound IV
(0.1% of R-R)
N677
Lymphocytosis18,0±2,1067,2±6,47**43,7±6,65 ° *
* the difference from the intact control; ° - contrast model
(° - P<0,05; * P<0,01; ** - P<0,001)

Table 6
The effect of compound IV on indicators cytogram (%) nasal rinse Guinea pigs
Group
The subpopulation
Intact control
n=6
Model
(rhinitis)
n=7
Compound IV
(0.1% of R-R)
n=7
Macrophages21,8±1,6412,7*±1,5714,6*±1,27
Lymphocytes4,7±0,886,1±1,425,7±1,54
Neutrophils0,3±0,213,3*±0,807,8**°±1,35
Eosinophils7,0±2,4547,9***±lower than the 5.376,9°°°±1,32
The epithelial cells66,2±3,0927,1***±5,0163,6°°°±3,02
* the difference from the intact control; ° - contrast model (rhinitis)
(° - P<0,05; **, °° - R<0,01; ***, °°° - P<0,001)

Table 7
The effect of compound IV on the absolute number of cell subpopulations nasal rinse Guinea pigs (1 ál)
Group
The subpopulation
Intact control
n=6
Model
(rhinitis)
n=7
Compound IV
(0,1-p)
n=7
Macrophages6,2±1,297,9±1,886,1±0,75
Lymphocytes0,9±0,244,1*±1,163,2*±52
Neutrophils0,03±0,0212,1±0,673,1±1,02
Eosinophils1,2±0,4626,5***±4,473,2°°±0,97
The epithelial cells11,8±1,411,3±2,1026,3**°°±or 4.31
Note: * - the difference from the intact control; ° - contrast model
(* - P<0,05; **,°° P<0,01; *** P<0,001)

From the data of tables 5-7, it follows that in terms of modeling allergic rhinitis compound IV significantly suppresses eosinophilic inflammation. This is confirmed by the reduction to normal absolute and relative number of eosinophils, as well as reduction of lymphocytosis in natalem flush.

Example 16

Anti-allergic activity of the compounds of General formula I on the model of allergic inflammation lay them the Guinea pigs

Used model of allergic lung inflammation in Guinea pigs.

Immunization of animals similar to that described in example 14.

24 hours after the last injection of ovalbumin produced the fence bronchoalveolar flushing (via a cannula inserted into the trachea), and changes in the bronchial mucosa was assessed by using a range of methods: histological and cytological.

The compounds (0.1% of R-R) was administered daily by inhalation using a nebulizer equipment for 6 days, on the 6th day of injection caused the provocation antigen (OA).

Table 8
The effect of compound IV on lymphocytosis (absolute number of cells in 1 μl) in bronchoalveolar washings
GroupIntact controlModel + provokes. (rhinitis)Compound IV
(0.1% of R-R)
N657
Lymphocytosis726,8±82,41849,4±287,3*1331,3±277,4
* P<0,01 - contrast to the intact control the I

Table 9
The effect of compound IV on the absolute number of cell subpopulations bronchoalveolar flushing Guinea pigs (1 ál)
Group
The subpopulation
Intact controlModelCompound IV
(0.1% of R-R)
Macrophages502,6±60,31
(n=6)
680,3±105,0
(n=5)
640,1±57,98
(n=6)
Lymphocytesof 101.4±24,3
(n=6)
328,3**±49,18
(n=5)
233,0*±45,77
(n=7)
Neutrophils0
(n=6)
56,8±17,08
(n=5)
16,9°±4,22
(n=7)
Eosinophils37,0±9,04
(n=5)
773,0**±171,7
(n=5)
487,4**±98,70
(n=6)
The epithelial cells16,9±6,09
(n=6)
0
(n=5)
2,48*±1,84
(n=7)
* - contrast INTA the private control; °- contrast model
(*,° - P<0,05; ** - P<0,01)

From the data of tables 8 and 9 it follows that the compound IV in the model of allergic inflammation of the lungs significantly inhibits the inflammatory process, which is a reduction of lymphocytosis, a decrease in the content key cells of allergic inflammation, eosinophils, a sharp decline in neutrophils and a decrease in the number of lymphocytes.

Example 17

The study of anti-inflammatory action of compounds of General formula I in models of inflammation of the lungs in rats induced by Sephadex

Model Sephadex-induced (6-day) pneumonia in rats

In experiments were used rats male Wistar breed weighing 270-300 g

Inflammation in the lungs induced single inhalation introduction of Sephadex A-25 (hydrophilic powder with a particle size of from 20 to 80 μm) in a dose of 5 mg/kg with a measuring device, which is the laboratory analogue inhaler "Ziklohaler" (research Institute of pulmonology of the RF).

The technique of inhalation of Sephadex and pharmacological substances

Rats with the original dosing device for inhalation dry powders under ether anesthesia was introduced Sephadex A-25 in the dose of 5 mg per 1 kg of body weight. Wistar rats after administration of Sephadex A-25 quickly come out and from anaesthesia and outwardly any peculiarities in the behavior, the nature of the breath they were not observed. Substances in the form of dry powder was introduced inhalation at a dose of 500 mcg/kg over 1 hour after administration of Sephadex, then for 5 consecutive days daily 1 time per day in the same morning. The control is represented by 2 groups: the group of intact animals, and a group of rats that inhaled once introduced Sephadex.

The results of therapeutic effects of pharmacological substances on the development of the inflammatory process in the lungs was assessed 6 days after aerosol exposure Sephadex using morphological and morphometric parameters (bulk density emphysema and alveolitis).

The methods used in the work

Histologic

Conducted histological examination of the lungs stained with hematoxylin and eosin.

Morphometric

Prepared histological sections of the lungs of a thickness of 4-5 microns, which counted the number of neutrophils millionary partitions, and estimated volumetric density of alveolitis and emphysema using grid Avtandilov (Avtandil - Introduction to quantitative pathological morphology. // - M.: Medicine, 1980, S. 203). Also conducted a morphometric study of the lymphoid tissue of the lungs. To this end, micropreparative lungs were fixed by the method inenstock et al. (Bienenstock J., Johnson N., Perey D.Y.E., Bronchiallymphoid tissue 1. Morphologic characteristics // Lab. Invest., 1973, v.28, p. 693-698). Lungs with trachea was removed from the thoracic cavity, and microreport were placed in a 2% aqueous solution of acetic acid. After 18-24 hours, the trachea, the main and lobar bronchi were cut off; the method of point account under a magnifying glass (×7) was performed morphometric assessment of bulk density of lymphoid tissue associated with the bronchi. Method point account determined bulk density of alveolitis and emphysema.

Cytological

Bronchoalveolar wash in rats and Guinea pigs received under geksenalovy anesthesia by twice washing the lungs through the trachea with 10 ml of saline. Cell viability was determined in the test with Trifanova blue. In bronchoalveolar fluid flush (BASS) with the camera Goryaeva determined the absolute number of cells in 1 ml (lymphocytosis). In smears of the sediment liquid BASS, obtained by centrifugation at 200 g for 10 minutes and then stained by Romanovsky-Giemsa, and counted andpulmonary they (in percent) [Avtsin A.P., Lukomsky GI, Romanov, L.K. et al. Andpulmonary they. // Sov. med., 1982, No. 7, S. 8-14].

The research results are processed by methods of variation statistics using t-student test.

Tables is 10
Indicators of bronchoalveolar lymphocytosis flushing of Wistar rats after aerosol exposure Sephadex A-25 and treatment with pharmacological agents (M±m)
Lymphocytosis
The absolute number of cells in 1 mm BALL
CriterionThe intact
n=5
The Sephadex
(model)
n=6
Compound IV
n=6
Lymphocytosisof 160.4±20,65259,2*±32,42178,6*±20,4
P0,050,05
Note: * - the difference from the intact control

Table 11
Indicators of cellular composition of bronchoalveolar flushing of Wistar rats after aerosol exposure Sephadex A-25 and the treatment of the compounds (M±m)
Lymphocytosis
The absolute number of cells of different subpopulations in 1 mm BALL
The subpopulationThe intact
control
Model
(Sephadex)
Compound IV
N456
Macrophagesof 124.8±16,35226,1*±30,83152,4±18,5
Lymphocytes15,5±4,2728,2±6,0122,0*±3,5
Neutrophils020,4*±6,385,1*±0,6
Note: * - the difference from the intact control (P<0,05)

Histological examination of lungs

Compound IV caused a distinct anti-inflammatory effect: the incidence of alveolitis was significantly lower compared with the model group of animals; emphysema is almost not detected; not marked infiltration of neutrophils millionary partitions. The level of lymphocytosis and the number of neutrophils in BAL inflammatory process is also much less pronounced than in animals treated with Sephadex within 5 days.

Thus, the whole complex used experimental models shows a significant antiallergic, antianaphylactic and anti-inflammatory AK is Yunosti compounds of General formula I, manifested in in vitro tests, and modeling, allergic and inflammatory pathology in vivo.

Example 18

The study of the hypolipidemic action of compounds of General formula (I) models of hypercholesterolemia in rats

The study was performed on rats male Wistar weighing 200±20 g Hyperlipidemia caused by oral administration of cholesterol load - oil suspension cholesterol:

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

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

Holt sodium (Sigma, USA), 100 mg/kg of body weight.

As the comparison drug used drug from the group of statins "mevacor" (lovastatin) Merck Sharp & Dohn at a dose of 40 mg/kg of Cholesterol suspension was injected in the morning daily for 10 days. The compounds (at a dose of 500 mcg/kg) and the reference product (at a dose of 40 mg/kg) was administered to animals with cholesterol suspension within 10 days. All animals received standard briquetted feed.

Animals were divided into following groups:

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

"Cholesterol" - rats treated per os cholesterol loading (n=10),

"Lovastatin" - rats treated per os cholesterol loading and lovastatin (n=10),

"compound IV" - rats treated per os cholesterol loading and investigational compound IV (n=10).

A blood sample of bralin 5, 8, day 10 of the experiment.

Statistical processing of the hypolipidemic action of the compounds under investigation conducted in relation to the group of "cholesterol" (table 13).

Table 12
Cholesterol and triglycerides in the serum and liver of rats treated for 10 days per os olive oil, cholesterol and compound IV simultaneously with cholesterol loading
Control
(n=6)
Cholesterol
(n=10)
Lovastatin
(n=10)
compound IV
(n=9)
Total cholesterol
Serum (mg/100 ml)67,2±6,1120,0±8,4100,5±6,7*to 96.6±5,7**
HDL
(mg/100 ml)
41,6±1,354,3±1,750,6±1,350,5±1,3*
LDL+VLDL (mg/100 ml)25,6±0,8 65,7±1,2to 49.9±0,9***46,1±0,9***
Liver
(mg/g tissue)
to 2.29±0,133,6±0,42,41±0,17***2,78±0,28***
Triglycerides
Serum (mg/100 ml)85,7±9,2to 99.2±8,793,6±7,595,1±7,9
Liver
(mg/g tissue)
3,89±0,146,83±0,396,25±0,13of 5.53±0,19***
* - p<0,1
** p<0,05
*** - p<0,01

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

Below are the results of other studies of the claimed compounds.

The experimental group included:

1) "Cholesterol" - treated for 10 days per os oil suspension of cholesterol,

2) "compound IV" - rats treated per os oil suspension of cholesterol and investigational compound IV,

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

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

5) connection V - 1Na" - rats treated per os oil suspension of cholesterol and analyzed monosodium salt of compound V,

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

7) "compound II - 1Na" - rats treated per os oil suspension of cholesterol and analyzed monosodium salt of compound II,

8) "Sim" - rats treated per os oil suspension of cholesterol and simvastatin,

9) control - intact rats before the start of the experiment.

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

In the serum was measured contents General holes the Wendy Erin, triglycerides, cholesterol, high density lipoprotein (HDL). Cholesterol of lipoproteins of low and very low density was calculated by the difference between total cholesterol and HDL cholesterol.

The content of total cholesterol and triglycerides in serum were determined by enzymatic method.

Determination of cholesterol in high density lipoprotein (α-PL) was performed by precipitation of LDL and lonp phosphorus-tungsten acid and magnesium ions.

Statistical processing

Data in tables are presented as mean ± standard error of the mean. The significance of differences between groups "cholesterol" and "the drug..." was assessed by two-sample t-test t-test. The probability of error (p) are indicated in the columns of the table.

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

Table 13
Experimental hypercholesterolemia
The lipid metabolism of rats (mg/100 ml)
before the start of the experiment (n=50)10 days of cholesterol on the power (n=20)
Total cholesterol
Serum59,9 ± 1,4135,7 ± 10,9
HDL38,2 ± 1,232,5 ± 1,1
LDL+lamp20,3 ± 1,3103,2 ± 11,5
Triglycerides
Serumof 83.6 ± 4,6of 156.6 ± 11,4
HDL19,0 ± 1,424,3 ± 1,8
LDL+VLDL58,7 ± 6,2to 132.6 ± 12,8

Ten rats to an oil suspension of cholesterol resulted in significant increase of total cholesterol in serum by 2.3 times, triglycerides 1.9 times. During the development of induced hyperlipidemia cholesterol HDL was reduced by 15%. Cholesterol LDL+VLDL increased in 5 times. Triglycerides LDL+lonp increased 2.3 times.

Table 14
Cholesterol and triglyceride levels (mg/100 ml) in the serum of rats on5 dayexperiment
IndicatorsGroup
"Cholesterol (n=20)"compound IV"
(n=20)
"compound IV-1Na"
(n=20)
"compound IV-2Na"
(n=19)
"Sim"
(n=10)
Total serum cholesterol92,3 ± 3,584,2 ± 3,972,0 ± 3,0
p = 0,0001
of 80.6 ± 2,8
p = 0.013
73,6 ± 7,3
p = 0.04
HDL34,3 ± 1,335,8 ± 1,233,1 ± 1,435,0 ± 1,327,8 ± 2,7
LDL+VLDL58,0 ± 4,148,5 ± 4,538,8 ± 2,9
p = 0,0006
44,3 ± 2,7
p = 0.02
43,0 ± 8,3
Triglyceridesserum 110,8 ± 7,1120,6 ± 8,9is 106.6 ± 6,2to 88.3 ± 4,8
p = 0.013
108,9 ± 6,9

Table 15
Cholesterol and triglyceride levels (mg/100 ml) in the serum of rats onDay 8experiment
IndicatorsGroup
"Cholesterol"
(n=20)
"compound IV"
(n=20)
"compound IV-1Na"
(n=20)
"compound IV-2Na"
(n=19)
"Sim"
(n = 10)
Total serum cholesterolto 127.2 ± 10,6103,3 ±8,1
p = 0,08
of 99.1 ± 6,0
p = 0.03
90,2 ± 5,1
p = 0.004
100,1 ± 12,8
HDL30,2 ± 1,035,9 ± 1,2
p = 0,0007
37,1 ± 1,4
p = 0,0003
35,9 ± 1,8
p = 0.01
33,2 ± 1,8
LDL+VLDL97,0 ± 11,367,3 ± 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
Serumto 150.6 ± 13,6to 154.2 ± 11,4146,4 ± 11,2for 125.8 ± 9,4119,9 ± 10,5
p = 0,085

Table 16
Cholesterol and triglyceride levels (mg/100 ml) in the serum of rats on10th dayexperiment
IndicatorsGroup
"Cholesterol"
(n=20)
"compound IV"
(n=20)
"compound IV-1Na"
(n=20)
"compound IV-2Na"
(n=19)
"Sim"
(n=10)
Total serum cholesterol135,7 ± 10,995,2 ± 5,4
p = 0.003
of 97.4 ± 7,1
p = 0.006
is 97.9 ± 3,9
p = 0.003
for 93.9 ± 10,2
p = 0.01
HDL32,5 ± 1,136,5 ± 0,9
p = 0,093
37,7 ± 1,0
p = 0.02
40,0 ± 1,9
p = 0.002
32,7 ± 1,2
LDL+VLDL103,2 ± 11,558,7 ± 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
Triglyceridesserumof 156.6 ± 11,4br143.3 ± 8,9132,5 ± 8,4
p = 0,096
RUB 127.3 ± 6,2
p = 0.03
142,0 ± 5,4

94,5 ± 9,2
Table 17
The development of hypercholesterolemia in experimental animals in the group "Cholesterol"
The lipid metabolism of rats (mg/10 ml)*
before the start of 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
Serumof 89.1 ± 1,8112,9 ± 9,2
p = 0,025
153,0 ± 14,7144,6 ± 12,8
HDL66,7 ± 1,147,5 ± 3,7
p < 0,02
50,3 ± 5,146,7 ± 2,9
LDL+VLDL22,5 ± 1,765,4 ± 10,6
p < 0,001
to 100.9 ± 17,3is 97.9 ± 14,0
Triglycerides
Serum74,8 ± 4,1129,7 ± 17,9115, 8mm ± 18,9
HDL43,1 ± 1,935,0 ± 3,1
p < 0,05
46,0 ± 4,635,5 ± 3,1
LDL+VLDL31,7 ± 3,759,5 ± 7,6
p < 0,05
76,0 ± 14,780,3 ± 16,2

Ten rats to an oil suspension of cholesterol resulted in significant increase of total cholesterol in serum 1.7 times, triglycerides 1.6 times (table 17). The HDL-cholesterol was decreased by 28% from the original 66,7 to 48 mg/100 ml during the development of induced hyperlipidemia. Cholesterol LDL+VLDL increased 4.4 times from 22.5 to 99 mg/100 ml

Table 18
Cholesterol and triglyceride levels (mg/100 ml) in the serum of rats on5 dayexperiment
IndicatorsGroup
"Cholesterol"
(n=12)
"compound IV-1Na"
(n=12)
"connection V-1Na"
(n=12)
"connecting the III-1Na"
(n=12)
"compound II-1Na"
(n=12)
Total serum cholesterol112,9 ± 9,294,4 ± 3,4
p = 0,079
to 96.9 ± 4,589,0 ± 5,0
p = 0,035
109,7 ± 9,4
HDL47,5 ± 3,760,7 ± 3,1
p = 0,012
55,1 ± 2,8
p = 0,11
49,0 ± 2,149,5 ± 3,8
LDL+VLDL65,4 ± 10,633,7 ± 4,5
p = 0.015
41,7 ± 6,5
p = 0,073
40,0 ± 5,4
p = 0,048
60,2 ± 10,6
Triglycerides
Serum94,5 ± 9,283,2 ± 6,363,3 ± 5,4
p = 0.009
87,4 ± 9,4of 80.9 ± 5,5
HDL35,0 ± 31 39,4 ± 3,530,9 ± 1,830,0 ± 2,232,6 ± 2,5
LDL+VLDL59,5 ± 7,643,7 ± 4,6
p = 0,09
32,4 ± 4,4
p = 0.007
57,4 ± 9,148,2 ± 4,9

Table 19
Cholesterol and triglyceride levels (mg/100 ml) in the serum of rats onDay 8experiment
IndicatorsGroup
"Cholesterol"
(n=12)
"compound IV-1Na"
(n=12)
"connection V-1Na"
(n=12)
the compound III-1Na"
(n=12)
"compound II-1Na"
(n=12)
Total cholesterol
Serum153,0 ± 14,7120,8 ± 4,3
p = 0.07
of 124.8 ± 5,8
p = 0,11
to 108.8 ± 8,2
p = 0.03
140,8 ± 13,6
HDL50,3 ± 5,154,9 ± 2,454,2 ± 3,448,4 ± 2,752,9 ± 4,1
LDL+VLDLto 100.9 ± 17,365,0 ± 6,0
p = 0,09
70,6 ± 6,8
p = 0,14
60,4 ± 8,3
p = 0.06
for 87.9 ± 14,5
Triglycerides
Serum129,7 ± 17,9116,3 ± 10,595,4 ± 5,7
p = 0,12
to 124.4 ± 8,9111,6 ± 17,2
HDL46,0 ± 4,645,8 ± 2,342,2 ± 2,145,2 ± 3,244,5 ± 2,5
LDL+VLDL76,0 ± 14,770,5 ± 9,053,2± 4,6 79,2 ± 7,773,9 ± 15,4

LDL+VLDL
Table 20
Cholesterol and triglyceride levels (mg/100 ml) in the serum of rats on10th dayexperiment
IndicatorsGroup
"Cholesterol"
(n=12)
"compound IV-1Na"
(n=12)
"connection V-1Na"
(n=12)
the compound III-1Na"
(n=12)
"compound II-1Na"
(n=11)
Total cholesterol
serum144,6 ± 12,8122,1 ± 9,8
p = 0,18
123,3 ± 7,3
p = 0,18
to 102.3 ± 6,9
p = 0.014
RUR 134.4 ± 12,5
HDL46,7 ± 2,953,4 ± 2,2
p = 0,09
52,9 ± 3,848,8 ± 2,454,7 ± 2,2
p = 0,046
is 97.9 ± 14,068,7 ± 10,1
p = 0,11
70,5 ± 8,9
p = 0,12
53,5 ± 7,3
p = 0.02
79,7 ± 12,6
Serum triglycerides115, 8mm ± 18,9to 109.3 ± 5,1to 81.9 ± 4,4
p = 0,11
to 109.3 ± 10,5to 108.5 ± 11,3
HDL35,5 ± 3,136,2 ± 2,631,5 ± 1,834,3 ± 2,338,9 ± 2,4
LDL+VLDL80,3 ± 16,273,1 ± 5,250,4 ± 4,8
p = 0.1
75,0 ± 8,569,6 ± 11,1

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

The results indicate that mono - and di-sodium salt was superior to compound IV by the dynamics of steps on total cholesterol is in the serum and LDL + VLDL and other lipid metabolism. While the 10-th day experience reliable and comparable decrease in these indicators occurred under the action of the aforementioned compounds and drug comparisons "Zocor (simvastatin), salts of compound IV was launched earlier experiment (5 and day 8). Both sodium salt of compound IV was increased HDL already to 5 days of the experiment, while compound IV was increased this figure is only 8 days. Comparison drug simvastatin had no effect on HDL-cholesterol. In addition, the disodium salt of compound IV reduced the levels of total serum triglycerides at 5 and 10 days of the experiment.

A distinctive property of the monosodium salt of compound V was the ability to reduce the levels of total serum triglycerides, while reducing total cholesterol, VLDL cholesterol and increased levels of HDL was lower than the monosodium salt of compound III and monosodium salt of compound IV.

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

Example 19

The study is gipolipidemicheskoe action of compounds of General formula (I) on the model of "endogenous" hypercholesterolemia Guinea pigs

The study was performed on Guinea pigs males (breed Agouti), initial mass 304±25 year duration of the experiment 31 days. The control group, 6 pigs - intact animals. The studied compounds were introduced from the first day of the experiment (from the first day of the introduction of the fat load).

Animals of the experimental groups every day for 31 days received per os investigated the connection and the fat load. Analyzed the connection in the following doses were administered in aqueous solution (0.5 ml per animal); fat load is a mixture of pork fat/pre-warmed corn oil, 4:1 by volume, at a rate of 5 ml/kg after 0.5 hours after administration of the tested substance.

Experimental group:

1) "control" - intact animals,

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

3) "compound IV" - the animals treated with the fat load + compound IV at a dose of 500 mcg/kg of body weight,

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

Data on the content of cholesterol and triglycerides in the serum of Guinea pigs treated with the fat load and the compounds presented in tables 22-25.

Table 21
The content of total cholesterol in serum cu is VI Guinea pigs, receiving fat load and the compounds
Total serum cholesterol
28 DN31 DN
Control37,4±3,3
Fat71,7±14,5
n=9
74,4±11,4
Compound IV55,5±6,3
n=10
49,2±3,9
P=0,064
Connection V52,9±6,5
n=9
46,3±4,1
P=0,043

Statistical data processing was performed using one-way ANOVA.

Table 22
The content of total triglycerides in the serum of Guinea pigs treated with the fat load and the compounds
Total serum triglycerides
28 DN31 DN
Control6,1±2,4
Fatof 89.7±14,0123,1±35,6
Compound IV66,8±6,769,0±13,4
Connection V62,3±5,456,0±6,1

Table 23
The total cholesterol content on the 31st day in the lipoprotein fractions of serum of Guinea pigs treated with the fat load and the compounds
Cholesterol, mmol/100 ml
TotalVLDLLDL
Control38,4±3,31,6±0,0732,2±2,8
Fat74,4±11,44,1±1,267,6±10,2
P=0.009
Compound IV49,2±3,93,0±0,7142,1±3,5
P=0,040
Connection V46,3±4,12,1±0,441,9±2,7
P=0,038

Table 24
The content of total triglycerides on the 31st day in the lipoprotein fractions of serum of Guinea pigs treated with the fat load and the compounds
Triglycerides, mg/100 ml
TotalVLDLLDL
Control60,1±2,3638,16±2,8616,13±0,97
Fatof 123.2±35,659,5±19,742,7±8,62
P=0.015
Compound IV69,0±13,433,2±12,329,6±2,4
Connection V56,0±6,1
P=0.1
22,5±4,4
P=0.1
23,1±1,9
P=0,053

The compounds IV and V were significantly reduced total cholesterol only by 31 day experiment 33.9 and 37.8%, respectively. However, they significantly reduced the LDL cholesterol 37.7%and 38%.

Figure 1.The change in total serum cholesterol under the influence of the fat load and different doses (50-1500 mg/kg) compound IV. Vertical thin lines specified standard deviation from the mean.

An advantage of the claimed compounds, in particular compounds IV is a wide range of effective doses that provides the breadth of its therapeutic action. For example, compound IV is almost equally effectively reduced the total cholesterol within 20 days in doses ranging from 50 to 1500 μg/kg, characterized in 30 times.

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

Example 20

A comparative study of the hypolipidemic action of compounds IV and glutamylcysteine (YY, compound III WO99/01103) in experimental models of hypercholesterolemia rats Wistar

Materials and methods

Presents data obtained after about what Yedinaya results of 3 experiments .

The study was performed on 118 rats male Wistar weighing 180-230 g Hyperlipidemia caused by oral administration (via stomach tube) cholesterol load - oil suspension cholesterol:

olive oil (refined, pre-heated 1 h 80aboutC) 5 ml/kg of body weight,

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

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

Cholesterol suspension was injected in the morning daily for 14 days. Animals received the compounds (at a dose of 500 mcg/kg of body weight) from day experience in the morning. All drugs were administered to the animals 30 minutes before cholesterol load.

Experimental group:

"Cholesterol" - rats treated for 14 days per os oil suspension cholesterol (n=37)

1. IV - rats treated per os oil suspension of cholesterol and Na salt of the compounds IV (n=37),

2. "YY" - rats treated per os oil suspension of cholesterol and investigated the connection YY/(n=22).

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

In serum measured total cholesterol, triglycerides, total cholesterol, high density lipoprotein (HDL). Cholesterol of lipoproteins of low and very low density is asciival on the difference between total serum cholesterol and HDL cholesterol.

The content of total cholesterol and triglycerides in serum were determined by enzymatic methods using test sets "CHOL L 250 S" (No. 10003268) and TG L 250 S" (No. 10003267) production company "Lachema (Czech Republic). The density measurement was performed on a spectrophotometer Shimadzu" at 500 nm, the calibration had to be attached to the test sets of standard samples.

Determination of cholesterol in high density lipoprotein (α-FP) was performed using test kits "HDL CHOL 250E" (10003202) and "CHOL L 250 S" (No. 10003267) firm "Lachema (Czech Republic) by precipitation of LDL and lonp phosphomevalonate acid and magnesium ions.

The data in tables 25-27 presented as average values ± standard error of the mean. The significance of differences between groups "cholesterol" and "the drug..." was assessed by two-sample t-test t-test. The probability of error (p) are listed in columns of tables.

Results studies

Experimental hypercholesterolemia

The initial level of total cholesterol and triglycerides in the blood plasma are presented in Table 25. Dynamics of changes of lipid metabolism in Table 26.

Table 25
The lipid metabolism of rats (n=24) before the start of the experiment
Initial biochemical parameters, mg/100 ml (n=30)
Total cholesterol
Serumto 72.3 ± 1,8
LHP36,1 ± 1,7
LDL+lamp36,2 ± 2,2
Cholesterol atherogenic index1,07 ± 0,05
Triglycerides
Serum69,0 ± 5,7
LHP15,4 ± 1,3
LDL+lamp53,6 ± 4,9
Triglyceride indexto 3.58 ± 0,22

The influence of the studied compounds on the development of hypercholesterolemia

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

Table 26
Change of parameters of lipid metabolism through experienceTotal cholesterol HDLsource5 days8 days10 days14 days"Cholesterol"36,1 ± 1,731,7 ± 1,627,3 ± 1,435,7 ± 1,529,9 ± 1,9GG26,4 ± 1,7
Phol-yy= 0,0323,5 ± 1,0
Phol-yy= 0,0632,2 ± 1,0
Phol-yy= 0,0425,4 ± 1,4
Phol-yy= 0,06IV33,3 ± 1,6
Pgg-IV= 0,00530,1 ± 1,4
Phol-IV= 0,07
Pgg-IV= 0,000341,6 ± 2,1
Phol-IV= 0,03
Pgg-IV= 0,000130,1 ± 1,6
Pgg-IV= 0,04Total cholesterol LDL"Cholestero the 36,2 ± 2,279,3 ± 4,776,3 ± 4,181,2 ± 6,9100,9* ± 9,5GG64,9 ± 3,1
Phol-yy= 0,0170,5 ± 2,961,0 ± 3,6
Phol-yy= 0,0165,3 ± 6,5
Phol-yy= 0,003IV69,2 ± 3,3
Phol-IV= 0,0866,9 ± 4,3
Phol-IV= 0,1265,8 ± 4,1
Phol-IV= 0,0669,2 ± 5,3
Phol-IV= 0,005Cholesterol atherogenic index (total cholesterol LDL+LOP/HDL)"Cholesterol"1,07 ± 0,052,9 ± 0,33,3 ± 0,32,7 ± 0,44,2 ± 0,7GG2,8 ± 0,33,1 ± 0,11,9 ± 0,1
Phol-yy= 0,062,7 ± 0,3
Phol-yy= 0,04IV2,3 ± 0,2
the hol-IV= 0,12,6 ± 0,3
Phol-IV= 0,07
Pgg-IV= 0,021,6 ± 0,1
Phol-IV= 0,008
Pgg-IV= 0,082,6 ± 0,3
Phol-IV= 0,03

Table 27
Significant (p ≤ 0,05) differences changes in parameters of lipid metabolism between groups IV and GG on a two-sample t-test t-test
Differences of changes of parameters of lipid metabolism in groups
daysGG (n=22)IV
Cholesterol HDL
5+ 22%
8+ 24%
10+ 26%
14+ 15,7%
Cholesterol atherogenic index (total cholesterol LDL+LOP/HDL)
5
8- 15%
10- 11%
Designation: - 10% - significantly below 10%. For 100% passed level in the group "Cholesterol".

Presented in tables 25-27 data clearly indicate that compound IV increases levels of cholesterol atherogenic HDL 10-14% compared with control, while under the influence of glutamylcysteine was significant lowering of HDL cholesterol.

As a result, the reduction of cholesterol atherogenic index by glutamylcysteine owing to lower cholesterol all fractions of plasma and HDL and LDL+lamp.

Compound IV differs from glutamylcysteine significant significant decrease in cholesterol atherogenic index by 20-40% during the experiment, due to the increase of the HDL fraction.

Comparison of lipid-lowering actions of glutamylcysteine with the appropriate activity IV showed the, that compound IV according to the invention is significantly superior to glutamylcysteine in raising HDL cholesterol and reducing the atherogenic index (table 27).

Example 21

Studies anti-inflammatory activity of compounds of General formula I (1% gel) to model carragenine edema in vivo

Materials and methods

Tests carried out on non-linear white mice weighing 30 to 32, Using the model carrageenin-induced edema by the method described in Winter et al. In: De Rosa M., Giroud J.P., D.A. Willoughby 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. In the right paw of the mouse subplantar impose a 1% solution carragenin (SERVA) in a volume of 0.05 ml of a Substance in a 1% gel applied on the paw 2 times: the first time immediately after the introduction carragenine; the second time is 1.5 hours. The measurement of the volume of the right and left (intact) paws carried out with the help of Vernier caliper after 3 h after injection carragenin. Inflammatory response and therapeutic effect of the impact estimate by the formula

where

P - gain and edema

On - volume feet after the introduction of logogen,

And - the value of the volume of the paws before the introduction of logogen.

The effect of therapeutic effects estimated by the degree of inhibition of the inflammatory response compared to the control and calculated by the formula

img src="https://img.russianpatents.com/1043/10438414-s.jpg" height="14" width="91" />

where

(o) - treated animals,

(K) - untreated animals.

Table 28
The effect of compounds of General formula I (1% gel) on the development of carragenine swelling of the paws of mice
AnalyteThe difference in the volume of pawIncrease (%)Inhibition of the inflammatory response (%)
Vin 0.104±0,009**60,043,8
V-1Na0,14±0,02**79,026,2
IV-1Na0,105±0,002**47,633,0
II-1Na0,1±0,009**32,351,6
III-1Na0,15±0,01*50,025,0
Voltaren0,08±0,006**25,861,3
* - statistically significant relative to the model * P<0,01
** P<0,01

The results of the experiments are presented in table 28 show a pronounced anti-inflammatory activity of the monosodium salt of compound (II), comparable with the activity of the comparison drug - voltaren.

Connection V and its monosodium salt show moderate activity in this model carragenine swelling of the paws of mice, inhibition of edema is 43.8% and 26.2%, respectively.

Thus, the whole complex used experimental models shows a significant antiallergic, antianaphylactic and anti-inflammatory activity of compounds of General formula I, as shown in in vitro tests, and modeling, allergic and inflammatory pathology in vivo. It is shown that the described compounds corresponding to General formula I show significant protective effect against systemic anaphylactic reactions in vivo, under conditions modeling of allergic rhinitis, in addition, these compounds suppress eosinophilic inflammation and have a distinct anti-inflammatory effect.

Examples of dosage forms

Example 22

A. Preformed shape

Tablet form is received, using the following ingredients:

The compound corresponding to General formula (I) or its pharmaceutically acceptable salt1-100 mgPotato starch20-50 mgMagnesium stearate3 mgAerosil1 mgLactoseup to 300 mg

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

B. Suppositories

An example of the composition of the suppository:

The compound corresponding to General formula (I) or its pharmaceutically acceptable salt1-100 mg
Cocoa butterthe amount needed to obtain suppository

If necessary, it is possible to manufacture rectal, vaginal and urethral suppositories with appropriate fillers.

Century Ointment

An example of the composition of the ointment:

The compound corresponding to General formula (I) or its pharmaceutically acceptable salt0,0-0,1 g
Vaseline10 g

Ointments are made by well-known technology.

, Gels

An example of the structure of the gel:

The compound corresponding to General formula (I) or its pharmaceutically acceptable salt1-100 mg
The carbopol200 mg
Benzyl alcohol : 20 mg
Ethanol300 mg
Waterto 10 g

D. Dry powder for inhalation

An example of the composition of the powder:

The compound corresponding to General formula (I) or its pharmaceutically acceptable salt0.2 g
Lactoseto 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:

The compound corresponding to General formula (I) or its farmaci is almost acceptable salt 1.5 to 150 mg
Purified waterto 15 ml

E. Eye drops

An example of the composition drops:

The compound corresponding to General formula (I) or its pharmaceutically acceptable salt0.5 to 50 mg
Preservative10 mg
Purified waterto 5 ml

E. injection

An example of the composition of the solution for injection:

The compound corresponding to General formula (I) or its pharmaceutically acceptable salt0.2-20 mg
Water for injection2 ml

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

where n is 2 or 3; and
R is

or their pharmaceutically acceptable salts, including the addition of glutaric anhydride or succinic acid in the form of solids to aqueous solution of amino acids of General formula

where R is a small town in the place

and, optionally, the transformation of the target product in its salt.

2. The method of obtaining the N-acyl derivatives of amino acids of General formula I

where n is 2 or 3; and
R is

or their salts, comprising conducting the reaction in a biphasic system of glutaric anhydride or succinic acid in a water-immiscible organic solvent with an aqueous solution of amino acids of General formula

or a salt thereof,
where R is

and, optionally, the transformation of the target product in its salt.



 

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25 cl, 1 tbl, 14 ex

FIELD: organic chemistry, medicine, hormones.

SUBSTANCE: invention describes imidazole derivatives of the formula (I) , racemic-diastereomeric mixtures and optical isomers, pharmaceutical salts wherein ---- represents an optional bond; R1 represents hydrogen atom (H), -(CH2)m-C(O)-(CH2)m-Z1, -(CH2)m-Z1; R2 represents hydrogen atom (H), or R1 and R2 are joined with nitrogen atoms to which they are bound forming compounds represented by formulae (Ia), (Ib) or (Ic) wherein R3 represents -(CH2)m-E-(CH2)m-Z2; R4 represents hydrogen atom (H) or -(CH2)m-A1; R5 represents (C1-C12)-alkyl, (C0-C6)-alkyl-C(O)-NH-(CH2)m-Z3 and optionally substituted phenyl; R6 represents hydrogen atom (H); R7 represents (C1-C12)-alkyl or -(CH2)m-Z4; m = 0 or a whole number from 1 to 6; n is a whole number from 1 to 5. Proposed compounds bind with subtypes of somatostatin receptors selectively.

EFFECT: valuable properties of compounds.

20 cl, 13776 ex

The invention relates to new imidazole derivative of the formula (I):where R1represents phenyl or pyridinyl, substituted by substituents selected from the group comprising (1) phenyl, (2) furyl, thienyl, (3) halogen, (4) halogen(lower)alkyl, (5) lower alkylthio, (6) nitro, (7) lower alkenyl, optionally substituted phenyl, (8) lower quinil, optionally substituted phenyl, (9) lower alkoxy, optionally substituted cyclo(lower)alkyl or phenyl, (10) lower alkyl, optionally substituted, phenyloxy or (11) amino, optionally substituted protected carboxyla; R2represents lower alkyl; R3represents halogen or lower alkyl; R4represents (1) lower alkenyl, optionally substituted phenyl, (2) phenyl, optionally substituted lower alkyl or lower alkenyl, (3) lower alkyl or (4) thienyl, optionally substituted with halogen; a represents a lower alkylene and L represents a simple bond, a lower albaniles or lower alkylene, optionally substituted phenyl or pyridinyl, or-X-CH2- where X represents O or NR5where R5represents hydrogen or n is

New drug substances // 2237657
The invention relates to organic chemistry and can find application in medicine

FIELD: chemistry.

SUBSTANCE: present invention refers to method of producing optically active monatin of identical configurations in positions 2 and 4 to be applied as sweetener. Isomerisation procedure implies that monatin of different configurations in positions 2 and 4 is isomerised in 2-nd position with aldehyde added in mixed solvent of water and organic solvent with pH 4 to 11. It is accompanied with simultaneous crystallisation of monatine with identical configurations in 2-position and 4-position.

EFFECT: development of effective method of producing optically active monatin isomers.

12 cl, 13 ex

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