RU2441061C2 - Docosahexaenoic acid derivatives and their application as drugs - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to new compounds of formula (I)

where X is carboxylic acid, carboxylates, carboxylic anhydride, diglyceride, triglyceride, phospholipid, or carboxamides, or to any their pharmaceutically acceptable salt. The invention particularly refers to (4Z, 7Z, 10Z, 13Z, 16Z, 19Z)-ethyl 2-ethyldocosa-4,7,10,13,16,19-hexanoate. The invention also refers to a food lipid composition and to a composition for diabetes, for reducing insulin, blood glucose, plasma triglyceride, for dislipidemia, for reducing blood cholesterol, body weight and for peripheral insulin resistance, including such compounds. Besides, the invention refers to methods for treating and/or preventing diabetes, dislipidemia, peripheral insulin resistance, body weight reduction and/or weight gain prevention, insulin, blood cholesterol, blood glucose and/or plasma triglyceride reduction.

EFFECT: higher clinical effectiveness.

61 cl, 4 tbl, 16 dwg, 5 ex

Description

FIELD OF THE INVENTION

The invention relates to compounds of the general formula (I):

and their use as medicines, in particular for the treatment of type 2 diabetes mellitus and prediabetic conditions. The present invention also relates to a pharmaceutical composition comprising compounds of formula (I), and to a fatty acid composition comprising compounds of formula (I).

BACKGROUND OF THE INVENTION

An increase in the incidence rate of type 2 diabetes worldwide is a huge medical problem and necessitates the introduction of successful preventive and therapeutic measures. An increase in the incidence of obesity and overweight, which strongly correlates with type 2 diabetes, negatively affects diabetes management and increases the likelihood of diseases associated with hypertension, dyslipidemia, and atherosclerosis.

The pathophysiological condition preceding the development of type 2 diabetes is associated with a reduced effect of insulin on peripheral tissues, this phenomenon is called insulin resistance. These tissues are mainly muscle, fatty tissue and liver tissue. In type 2 diabetes, insulin resistance is most characteristic of muscle tissue. A syndrome in which insulin resistance, high blood pressure, dyslipidemia, and a pro-inflammatory condition are observed is called metabolic syndrome. The prevalence of metabolic syndrome in adults in developed countries is 22-39% (Meighs 2003).

Currently, the most promising way to reduce the metabolic syndrome is to change the lifestyle, which consists in reducing weight, reducing the intake of saturated fat, increasing physical activity in combination with appropriate pharmacotherapy. With healthy diets that avoid excessive energy intake, saturated fats are replaced with mono- and polyunsaturated fatty acids. In the prevention of type 2 diabetes, omega-3 polyunsaturated fatty acids found in fatty fish, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have proven particularly effective.

EPA and DHA influence various physiological processes, such as regulation of plasma lipids, cardiovascular and immune functions, insulin action, development of the nervous system and visual function. There is evidence of the role of these acids in the prevention and correction of coronary heart disease, dyslipidemia, type 2 diabetes, insulin resistance and hypertension (Simonopoulos 1999; Geleijnse 2002; Storlien 1998). Recent studies have shown that omega-3 polyunsaturated fatty acids are important mediators of gene expression, acting through nuclear receptors such as peroxisome proliferation activating receptors (PPARs), controlling the expression of genes related to carbohydrate and lipid metabolism and lipogenesis (Jump 2002) . Receptors PPARs are nuclear receptors that play an important role in the development of obesity-related metabolic disorders, such as hyperlipidemia, insulin resistance and coronary artery disease.

Three receptor subtypes, α, γ and δ, have different expression patterns and recognize the components of different lipoproteins, and regulate lipid homeostasis based on the needs of specific tissues. PPARα enhances the catabolism of fatty acids in the liver and is a molecular target for lipids that lower lipids. PPARγ plays a role in adipocyte differentiation and mediates the activity of insulin sensitizers - thiazolidinediones (glitazones), the mechanisms of this mediation are poorly understood (Chih-Nao 2003; Yki-Jarvinen 2004).

Recently, drugs acting as PPARγ receptor ligands have been used in the treatment of type 2 diabetes (Yki-Jarvinen 2004). These compounds, called thiazolidinediones or glitazones, are drugs that reverse insulin resistance, which is the pathophysiological basis for the development of metabolic syndrome and type 2 diabetes. These compounds, from which rosiglitazone and pioglitazone are available as drugs, lower the fasting glucose concentration and the postprandial glucose concentration (as shown by the glucose tolerance test), plasma insulin levels, and free fatty acids. In this regard, glitazones act as insulin sensitizers.

However, these improvements are usually accompanied by weight gain and an increase in the mass of subcutaneous adipose tissue (Adams 1997). The use of thiazolidinediones is not only associated with weight gain, but some patients also experience fluid retention and an increase in plasma volume, which leads to peripheral edema. Weight gain and swelling can lead to impaired cardiac function, which is why the Food and Drug Administration (FDA) included a warning in the information on rosiglitazone (Avandia) and pioglitazone (Takeda). These adverse effects limit the use of glitazones, especially in patients with coronary heart disease. Obviously, there is a need to develop new drugs that have a positive effect on insulin resistance, but reduce body weight and do not contribute to water retention in the body.

The effect of polyunsaturated fatty acids (PUFAs) on PPARs is not only the result of fatty acid structure and receptor affinity. Factors affecting the concentration of non-esterified fatty acids (NEFA) are also important. The NEFA pool is affected by the concentration of exogenous fatty acids entering the cell, and the amount of endogenous synthesized fatty acids, their removal through incorporation into lipids, and their oxidation pathways (Pawar 2003).

Although omega-3 fatty acids are weak PPARs agonists compared to pharmacological agonists such as thioglitazones, these fatty acids have shown improved glucose uptake and increased insulin sensitivity (Storlien 1987). It was reported that adipocytes were more sensitive to insulin and transported more glucose when the ratio of polyunsaturated fatty acids to saturated fatty acids was increased in the diet (Field 1990). Together, these data indicate that C20 and C22 fatty acids, called EPA and DHA, can play a role in preventing the development of insulin resistance.

Due to their limited in vivo stability and lack of biological specificity, PUFAs are not truly widely used therapeutic agents. Several research groups have carried out chemical modifications of p-3 polyunsaturated fatty acids in order to change or increase their metabolic effects.

For example, the lipid-lowering effect of EPA was enhanced by introducing methyl or ethyl at the α- or β-position of EPA (Vaagenes 1999). These compounds also reduce plasma free fatty acids, while EPA ethyl ester does not.

In a recent paper published by L. Larsen (Larsen 2005), the authors show that α-methyl derivatives of EPA and DHA increase the activation of the nuclear receptor PPARα and thus the expression of L-FABP compared to EPA / DHA. EPA with an ethyl group in the α-position activates PPARα with the same strength as α-methyl-EPA. The authors suggest that the delayed catabolism of these α-methyl fatty acids may contribute to their increased effects due to reduced β-oxidation in mitochondria, leading to peroxisomal oxidation.

Alpha-methyl-EPA has been shown to be a more potent inhibitor of platelet aggregation than EPA, both in vitro (Larsen 1998) and in vivo (Willumsen 1998). In the abstract of Japanese patent publication number 05-00974, DHA is substituted at the alpha position with an OH group, but only as an intermediate. No studies of the possible pharmaceutical effects of this compound have been reported.

Laxdale Limited has also described the use of alpha-substituted EPA derivatives in the treatment of psychiatric disorders and disorders of the central nervous system (US 6689812).

None of these modified fatty acids, however, had satisfactory pharmaceutical activity and none of them entered the pharmaceutical market.

SUMMARY OF THE INVENTION

An object of the present invention is to provide novel derivatives of DHA having a therapeutic effect.

Based on the present invention, a number of aspects are presented in the appended claims. Some of these aspects are the following aspects:

1. New compounds, ie some α-substituted polyunsaturated fatty acid derivatives.

2. New compounds for use as medicines and for use in therapy.

3. A fatty acid composition or a pharmaceutical composition comprising new compounds.

4. A fatty acid composition comprising new compounds for use as a medicine and for use in therapy.

5. The use of new compounds to obtain drugs for the prevention and / or treatment of diabetes in humans or animals.

6. The use of new compounds to obtain drugs for the treatment and / or prevention of obesity or overweight.

7. The use of new compounds to obtain drugs for controlling weight loss and / or for the prevention of weight gain.

8. The use of new compounds for the manufacture of a medicament for the treatment and / or prevention of diseases associated with amyloidosis.

9. The use of new compounds for the manufacture of a medicament for the treatment or prophylaxis of multiple risk factors for cardiovascular diseases.

10. The use of new compounds for the manufacture of a medicament for the prevention of cerebrovascular accident, cerebral or transient ischemic attacks associated with atherosclerosis of certain arteries.

11. A method for the specific treatment of a diabetic disease, preferably type 2 diabetes.

12. A method of controlling weight loss, preventing weight gain and / or treating and / or preventing obesity or overweight.

13. A method for the treatment and / or prevention of diseases associated with amyloidosis

14. A method for the treatment or prevention of multiple risk factors for cardiovascular disease.

15. A method for the prevention of cerebrovascular accidents, cerebral or transient ischemic attacks associated with atherosclerosis of certain arteries.

16. Methods of obtaining new analogues of fatty acids corresponding to the present invention

The present invention relates to a compound of formula (I):

where - R ₁ and R ₂ are the same or different and can be selected from the group consisting of a hydrogen atom, a hydroxyl group, an alkyl group, a halogen atom, an alkoxy group, an acyloxy group, an acyl group, an alkenyl group, an alkynyl group, an aryl group, an alkylthio group, alkoxycarbonyl group, alkylsulfinyl group, alkylsulfonyl group, amino group, alkylamino group; and

- X means a carboxyl group, a carboxylate group or a carboxamide group or any pharmaceutically acceptable salt, solvate, complex or prodrug thereof, provided that:

- the compound of formula (I) is not (completely Z) -4,7,10,13,16,19-docosahexaenoic acid (DHA), alpha-methyl-DHA, methyl ether alpha-methyl-DHA, ethyl ether alpha methyl DHA or ethyl ester alpha-hydroxy-DHA.

The conditions apply to the following cases:

- when R ₁ is a hydrogen atom, R _{2 is} not a hydrogen atom;

- when R ₂ is a hydrogen atom, R _{1 is} not a hydrogen atom;

- when R ₁ is a methyl group, R _{2 is} not a hydrogen atom and X is not a carboxyl group, methyl carboxylate or ethyl carboxylate;

when R ₂ is a methyl group, R _{1 is} not a hydrogen atom, and X is not a carboxyl group, methyl carboxylate or ethyl carboxylate;

- when R ₁ is a hydroxyl group, R _{2 is} not a hydrogen atom and X is not ethyl carboxylate; and

- when R ₂ is a hydroxyl group, R _{1 is} not a hydrogen atom and X is not ethyl carboxylate.

In the compound of the present invention, said alkyl group may be selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, n-hexyl and benzyl groups; said halogen atom may be selected from the group consisting of fluorine, chlorine, bromine and iodine; said alkoxy group may be selected from the group consisting of methoxy, ethoxy, propoxy, isopropoxy, sec-butoxy, phenoxy, benzyloxy, OCH ₂ CF ₃ and OCH ₂ CH ₂ OCH ₃ groups; said acyloxy group may be selected from acetoxy, propionoxy and butyroxy groups; said alkenyl group may be selected from the group consisting of allyl, 2-butenyl and 3-hexenyl groups; said alkynyl group may be selected from the group consisting of propargyl, 2-butynyl and 3-hexenyl groups; said aryl group is a phenyl group; said alkylthio group may be selected from the group consisting of methylthio, ethylthio, isopropylthio and phenylthio; said alkoxycarbonyl group may be selected from the group consisting of methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl and butoxycarbonyl groups; said alkylsulfinyl group may be selected from the group consisting of methanesulfinyl, ethanesulfinyl and isopropanesulfinyl groups; said alkylsulfonyl group may be selected from the group consisting of methanesulfonyl, ethanesulfonyl and isopropanesulfonyl groups; said alkylamino group may be selected from the group consisting of methylamino, dimethylamino, ethylamino and diethylamino groups; said carboxylate group may be selected from the group consisting of ethyl carboxylate, methyl carboxylate, n-propyl carboxylate, isopropyl carboxylate, n-butyl carboxylate, sec-butyl carboxylate and n-hexyl carboxylate; said carboxamide group may be selected from the group consisting of primary carboxamide, N-methylcarboxamide, N, N-dimethylcarboxamide, N-ethylcarboxamide and N, N-diethylcarboxamide groups.

In one embodiment of the present invention, R ₁ and R ₂ are selected from the group consisting of a hydrogen atom, a hydroxy group, an alkyl group, a halogen atom, an alkoxy group, an alkylthio group, an alkylsulfinyl group, an alkylsulfonyl group, an amino group and an alkylamino group.

In another embodiment of the present invention, R ₁ and R ₂ are selected from the group consisting of a hydrogen atom, a hydroxy group, a C ₁ -C ₇ alkyl group, a halogen atom, a C ₁ -C ₇ alkoxy group, a C ₁ -C ₇ alkylthio group, C ₁ - A C ₇ alkylsulfinyl group, a C ₁ -C ₇ alkylsulfonyl group, an amino group and a C ₁ -C ₇ alkylamino group. Said C ₁ -C ₇ alkyl group may be a methyl, ethyl or benzyl group; said halogen atom may be fluorine or iodine: said C ₁ -C ₇ alkoxy group may be a methoxy or ethoxy group; said C ₁ -C ₇ alkylthio group may be methylthio, ethylthio or phenylthio; said C ₁ -C ₇ alkylsulfinyl group may be an ethanesulfinyl group; said C ₁ -C ₇ alkylsulfonyl group may be an ethanesulfonyl group; said C ₁ -C ₇ alkylamino group may be an ethylamino or diethylamino group; and X may be an ethyl carboxylate or carboxamide group.

In another embodiment of the present invention, R ₁ and R ₂ are selected from the group consisting of a hydrogen atom, a C ₂ -C ₇ alkyl group, a halogen atom, a C ₁ -C ₇ alkoxy group, a C ₁ -C ₇ alkylthio group, a C ₁ -C ₇ an alkylsulfinyl group, a C ₁ -C ₇ alkylsulfonyl group, an amino group and a C ₁ -C ₇ alkylamino group; and X is carboxylate. Said C ₂ -C ₇ alkyl group may be an ethyl or benzyl group; said halogen atom may be fluorine or iodine; said C ₁ -C ₇ alkoxy group may be a methoxy or ethoxy group; said C ₁ -C ₇ alkylthio group may be methylthio, ethylthio or phenylthio; said C ₁ -C ₇ alkylsulfinyl group may be an ethanesulfinyl group; said C ₁ -C ₇ alkylsulfonyl group may be an ethanesulfonyl group; said C ₁ -C ₇ alkylamino group may be an ethylamino or diethylamino group; and X may be ethyl carboxylate.

In the compound corresponding to formula (I) of the present invention, R ₁ and R ₂ may be the same or different. When they are different, the compounds of formula (I) are able to exist in stereoisomeric forms. This is understood in such a way that the present invention includes all optical isomers of the compounds of formula (I) and mixtures thereof, including racemates.

Therefore, where R ₁ is different from R ₂ , the present invention includes compounds of formula (I) which are racemic or enantiomerically pure, or as (S) or (R) enantiomer. Therefore, where R ₁ is different from R ₂ , the present invention includes compounds of formula (I) that are racemic or enantiomerically pure, or as a (S) or (R) stereoisomer.

Within the scope of the present invention are enantiomers of the compounds of formula (I) as defined above. In addition, the enantiomers of the DHA derivatives of the present invention may be in the form of a carboxylic acid or a pharmaceutically acceptable salt thereof, any ester, anhydride or amide (primary, secondary, tertiary). The acid derivative may be in the form of a phospholipid or tri-, di- or monoglyceride.

In one embodiment of a compound of formula (I) according to the present invention, one of R ₁ and R ₂ is a C ₂ -C ₇ alkyl group, for example ethyl or benzyl, and the other is a hydrogen atom. Preferably, the alkyl group is ethyl.

In another embodiment of a compound of formula (I) according to the present invention, one of R ₁ and R ₂ is an alkoxy group, for example an ethoxy or methoxy group, and the other is a hydrogen atom.

In another embodiment of a compound of formula (I) according to the present invention, one of R ₁ and R ₂ is a halogen atom, for example fluorine or iodine, and the other is a hydrogen atom.

In another embodiment of a compound of formula (I) according to the present invention, one of R ₁ and R ₂ is an alkylthio group, for example an ethylthio, methylthyl or phenylthio group, and the other is a hydrogen atom. Preferably, the alkylthio group is an ethylthio group.

In another embodiment of a compound of formula (I) according to the present invention, one of R ₁ and R ₂ is an alkylsulfonyl group, for example ethylsulfonyl, and the other is a hydrogen atom.

In another embodiment of a compound of formula (I) according to the present invention, one of R ₁ and R ₂ is an amino group, and the other is a hydrogen atom.

In another embodiment of a compound of formula (I) according to the present invention, one of R ₁ and R ₂ is an alkylamino group, for example an ethylamino or diethylamino group, and the other is a hydrogen atom.

In a further embodiment of the compound of formula (I) according to the present invention, R ₁ and R ₂ are the same and are C ₁ -C ₇ alkyl groups, preferably methyl or ethyl groups.

In preferred embodiments of the compound of formula (I), X is a carboxylate, for example ethyl carboxylate.

The compound of the present invention may exist in the form of a phospholipid, tri-, di- or monoglyceride, or in the form of a free acid.

The alpha substituted DHA derivatives of the present invention were very active pharmaceutically. In particular, the fatty acid derivatives of the present invention have great potential for use in the treatment and / or prophylaxis of diabetes and pre-diabetic conditions.

Another aspect of the present invention relates to a compound of formula (I) for use as a medicine.

The present invention also relates to a method for producing a compound of formula (I). For example, a compound of formula (I) can be prepared from (all-Z) -4,7,10,13,16,19-docosahexaenoic acid (DHA). DHA can be of plant, microbial and / or animal origin (for example, marine fish fat). Another important advantage with the compounds of formula (I) is that fatty acid analogs can be obtained directly on the basis of (full-Z) -4,7,10,13,16,19-docosahexaenoic acid (DHA).

In a preferred embodiment of the present invention, fatty acid analogs of formula (I) are derived from DHA, wherein said DHA is derived from at least one source of plant, microbial and animal origin, or a combination thereof. The present invention therefore includes derivatives derived from fat of microbial origin containing DHA. Said DHA is derived from sea fat, such as fish oil.

Another aspect of the present invention relates to a pharmaceutical composition comprising a compound of formula (I) as an active ingredient. The pharmaceutical composition may include a pharmaceutically active carrier. Accordingly, the pharmaceutical composition of the present invention is formulated for oral administration, for example, in the form of a capsule or sachet powder. An appropriate daily dosage of a compound of formula (I) according to the present invention is from 10 mg to 10 g, in particular from 100 mg to 1 g of said compound.

In addition, the present invention relates to a fatty acid composition comprising a compound of formula (I). At least 60% or at least 90% of the weight of the fatty acid composition may comprise the compound. The fatty acid composition may further include (all-Z) -5,8,11,14,17-eicosapentaenoic acid (EPA), (all-2) -4,7,10,13,16,19-docosahexaenoic acid (DHA ), (all-Z) -6,9,12,15,18-gene-capase-pentaenoic acid (HPA), and / or (all-Z) -7,10,13,16,19-docosapentaenoic acid (DPA). Fatty acids may be present in the form of derivatives. The fatty acid composition of the present invention may further include a pharmaceutically acceptable antioxidant, for example, tocopherol. Also within the scope of the present invention is the fatty acid composition described above for use as a medicine.

In an additional aspect, the present invention relates to the use of a compound according to formula (I) for the manufacture of a medicament for controlling weight loss and / or preventing weight gain; for the manufacture of a medicament for the treatment and / or prevention of obesity or overweight; for the manufacture of a medicament for the prevention and / or treatment of diabetes in animals, especially type 2 diabetes; for the manufacture of a medicament for the treatment and / or prevention of diseases associated with amyloidosis; for the manufacture of a medicament for the treatment or prevention of multiple risk factors for cardiovascular diseases, especially for the treatment of elevated blood lipids, for the manufacture of a medicament for the prevention of cerebrovascular accident, cerebral or transient ischemic attacks associated with atherosclerosis of certain arteries.

In addition, the present invention relates to a method for controlling weight loss and / or preventing weight gain; a method for the treatment and / or prevention of obesity or overweight; a method for the prevention and / or treatment of diabetes, especially type 2 diabetes; a method for treating and / or preventing diseases associated with amyloidosis; a method for treating or preventing multiple cardiovascular disease risk factors; a method for the prevention of cerebrovascular accident, cerebral or transient ischemic attacks associated with atherosclerosis of certain arteries, where a pharmaceutically effective amount of a compound of formula (I) is administered to a human or animal. Accordingly, the compound of formula (I) is administered orally in humans or animals.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic description of the theory of a pool of free fatty acids.

The figure 2 presents a description of the models and methods used in the present invention to demonstrate the effects on metabolic syndrome and type 2 diabetes.

The figure 3 shows the concentration of free fatty acids of various compounds corresponding to the present invention, in the liver tissue of animals treated with these compounds at a concentration of 1.5% of the total fat content.

The figure 4 shows the intracellular concentration of DHA in the liver tissue of animals that received various compounds corresponding to the present invention, at a concentration of 1.5% of the total fat content.

Figure 5 shows the binding affinity for the PPARγ receptor of various compounds of the present invention.

Figure 6 shows the binding affinity for the PPARα receptor of various compounds of the present invention.

Figure 7 shows the binding affinity for the RXRα receptor of various compounds of the present invention.

Figure 8 shows the release of luciferase from transfected cells treated with various compounds of the present invention,

The figure 9 shows the experimental plan of block 4.

The figure 10 shows the change in body weight during 2 weeks of the experimental diet after 8 weeks of a diet high in fat.

11 shows the results of luciferase activity, i.e. endogenous activity of PPARγ.

Figure 12 shows endogenous luciferase activity with various compounds of the present invention compared to DHA.

Figure 13 shows a typical blood glucose decrease curve before and after animals with insulin resistance were given a compound that decreased insulin resistance.

Figures 14, 15 and 16 show various effects of the DHA derivatives of the present invention on metabolic syndrome and insulin resistance.

DETAILED DESCRIPTION OF THE INVENTION

In the research work leading up to the present invention, new DHA derivatives were obtained that showed high pharmaceutical activity.

Fatty acids penetrate the cell passively or through G-protein conjugated transport systems, such as transport proteins of fatty acids. Inside cells, they are temporarily bound by binding proteins (fatty acid binding proteins, FABPs), which play an important role in directing fatty acids to different places in the cell for metabolism and gene expression (Pawar & Jump 2003). (Figure 1 liver cell).

The esterification of fatty acids into triglycerides, polar lipids, cholesterol esters and their beta oxidation (mitochondrial and peroxisomal) requires the conversion of fatty acids to acyl CoA thioesters. Other metabolic pathways, such as microsomal NADPH-dependent monoxidation and eicosanoid synthesis, use unesterified fatty acids as substrates. All these reactions, apparently, affect the concentration of free fatty acids (unesterified) in the cell and, thus, the amount and type of fatty acids that can be used as ligands for nuclear receptors. Since PPARs are known to bind to non-esterified fatty acids, it seems reasonable to assume that the composition of the fatty acid pool is an important factor in PPAR activity.

The composition of the pool of free fatty acids is affected by the concentration of exogenous fatty acids entering the cells and the rate of their removal through the metabolic pathways listed above. Since short and medium chain fatty acids are required for these metabolic pathways, practically only polyunsaturated fatty acids with long chains will be free to bind to nuclear receptors. In addition, the structure of a fatty acid may also be an important factor. Even if a series of mono- and polyunsaturated fatty acids showed affinity for the PPARα receptor, EPA and DHA showed the highest binding ability in experiments with rat liver cells (Pawar & Jump 2003).

The search for fatty acid candidates available for genetic modification of proteins by interacting with nuclear receptors like PPARs is important in order to confirm that the corresponding fatty acids will enrich the pool of free fatty acids.

DHA, which enters the cells, is rapidly converted to acyl-CoA thioesters and incorporated into phospholipids, as a result of which the intracellular concentration of DHA is relatively low. These DHA-CoA are also a substrate for β-oxidation initially in peroxisomes, which leads to retroconversion of DHA EPA, see figure 1. Due to the rapid incorporation into neutral lipids and oxidation, DHA will not linger for a long time in the pool of free fatty acids. As a consequence, the effect of DHA on gene expression is probably limited.

The present invention aims to accumulate derivatives of fatty acids in a pool of free fatty acids rather than incorporation into phospholipids. The inventors were surprised to find that the introduction of at least one substituent at the α-position of DHA results in a lower oxidation rate and less inclusion in neutral lipids. This leads to an increased effect on gene expression, since DHA derivatives will accumulate in the tissues of the liver, muscle tissue, fat cells and stimulate the activity of nuclear receptors to a greater extent than DHA.

The various substituents of the present invention will give varying degrees of affinity for derivative derivatives. It is also possible that changes in the affinity for fatty acid binding proteins lead to changes in the biological activity of these α-substituted DHA derivatives of formula (I). In general, these changes lead to an increase in the therapeutic effect of the DHA derivatives of the present invention compared to DHA.

EPA (all-Z) -5,8,11,14,17-eicosapentaenoic acid) was previously alkylated at the α- and β-position in order to inhibit the mitochondrial β-position. DHA is not oxidized in mitochondria, but is incorporated into phospholipids. In peroxisomes, some DHA is converted to EPA. The substituent at the α-position of EPA and DHA will affect various metabolic pathways. It has been shown previously that α-methyl-EPA and β-methyl-EPA are included in phospholipids and triglycerides, while α-ethyl-EPA is not (Larsen 1998). In this study, derivatives were tested as substrates and / or inhibitors of enzymes that play a role in the cascade of eicasanoids. Since most of the substrates for these enzymes are fatty acids freed from phospholipids, it is desirable that derivatives be included in phospholipids. In contrast, as mentioned above, we wanted the derivatives not to be included in lipids, but to accumulate in the NEFA pool.

In this description, the abbreviation "PRB-x", where x is an integer, will be used to describe the specific compounds of the present invention. The structural formulas and common names of these compounds are given below.

PRB-1 α-methyldocosahexaenoic acid ethyl ester

PRB-2 ethyl ester of α-ethyl docosahexaenoic acid

PRB-3 α-ethoxydocosahexaenoic acid ethyl ester

PRB-4 α-fluorodocosahexaenoic acid ethyl ester

PRB-5 α, α-dimethyldocosahexaenoic acid ethyl ester

PRB-6 α-thiomethyldocosahexaenoic acid ethyl ester

PRB-7 α-thioethyl docosahexaenoic acid ethyl ester

PRB-8 α, α-diethyl docosahexaenoic acid ethyl ester

PRB-9 α-benzyldocosahexaenoic acid ethyl ester

PRB-10 α-ethanesulphenyldocosahexaenoic acid ethyl ester

PRB-11 α-thiophenyl docosahexaenoic acid ethyl ester

PRB-12 α-hydroxydocosahexaenoic acid ethyl ester

PRB-13 α-methyldocosahexaenoic acid amide

PRB-14 α-methoxydocosahexaenoic acid ethyl ester

PRB-15 ethyl ester of α-iododocosahexaenoic acid

PRB-17 α-aminodocosahexaenoic acid ethyl ester

PRB-18 (4R, 5S) -3-docosahexaenoyl-4-methyl-5-phenyl-oxazolidin-2-one

PRB-19 (4R, 5S) -3 - [(S) -α-ethyldocosahexaenoyl] -4-methyl-5-phenyl-oxazolidin-2-one

PRB-20 ethyl ester of (S) - (+) - α-ethyldocosahexaenoic acid

PRB-21 (4S, 5R) -3-docosahexaenoyl-4-methyl-5-phenyl-oxazolidin-2-one

PRB-22 (4S, 5R) -3 - [(R) -α-ethyldocosahexaenoyl] -4-methyl-5-phenyl-oxazolidin-2-one

PRB-23 ethyl ester of (R) - (-) - α-ethyldocosahexaenoic acid

PRB-24 2- (1,3-dioxo-1,3-dihydro-isoindol-2-yl) -docosahexaenoic acid ethyl ester

PRB-25 α-ethylaminodocosahexaenoic acid ethyl ester

PRB-26 α-diethylamino-docosahexaenoic acid ethyl ester

PRB-1 corresponds to a compound of formula (I), wherein R ₁ or R ₂ is methyl and the second is hydrogen and X is ethyl carboxylate.

PRB-2 corresponds to a compound of formula (I), wherein R ₁ or R ₂ is ethyl and the second is hydrogen and X is ethyl carboxylate.

PRB-3 corresponds to a compound of formula (I), wherein R ₁ or R ₂ is ethoxy and the second is hydrogen and X is ethyl carboxylate.

PRB-4 corresponds to a compound of formula (I), wherein R ₁ or R ₂ is fluoro and the other is hydrogen and X is ethyl carboxylate.

PRB-5 corresponds to the compound of formula (I), wherein R ₁ and R _{2 are} methyl and X is ethyl carboxylate.

PRB-6 corresponds to a compound of formula (I), wherein R ₁ or R ₂ is methylthio and X is ethyl carboxylate.

PRB-7 corresponds to a compound of formula (I), wherein R ₁ or R ₂ is an ethylthio group and the other is hydrogen, and X is ethyl carboxylate.

PRB-8 corresponds to a compound of formula (I), wherein R ₁ and R _{2 are} ethyl and the other is hydrogen and X is ethyl carboxylate.

PRB-9 corresponds to a compound of formula (I), wherein R ₁ or R ₂ is benzyl and the other is hydrogen and X is ethyl carboxylate.

PRB-10 corresponds to a compound of formula (I), wherein R ₁ or R ₂ is ethanesulfinyl and the other is hydrogen and X is ethyl carboxylate.

PRB-11 corresponds to a compound of formula (I), wherein R ₁ or R ₂ is phenylthio and the other is hydrogen and X is ethyl carboxylate.

PRB-12 corresponds to a compound of formula (I), wherein R ₁ or R ₂ is hydroxy and the other is hydrogen, and X is ethyl carboxylate.

PRB-13 corresponds to a compound of formula (I), wherein R ₁ or R ₂ is methyl and the other is hydrogen, and X is primary carboxamide.

PRB-14 corresponds to a compound of formula (I), wherein R ₁ or R ₂ is methoxy and the other is hydrogen and X is ethyl carboxylate.

PRB-15 corresponds to a compound of formula (I), wherein R ₁ or R ₂ is iodo and the other is hydrogen and X is ethyl carboxylate.

PRB-17 corresponds to a compound of formula (I), wherein R ₁ or R ₂ is an amino group and the other is hydrogen, and X is ethyl carboxylate.

PRB-20 corresponds to the (S) stereoisomer of the compound of formula (I), wherein R ₁ or R ₂ is ethyl and the other is hydrogen and X is ethyl carboxylate.

PRB-23 corresponds to the (R) stereoisomer of a compound of formula (I), wherein R ₁ or R ₂ is ethyl and the other is hydrogen and X is ethyl carboxylate.

PRB-24 corresponds to a compound of formula (I), wherein R ₁ or R ₂ is N-phthalimide and the other is hydrogen, and X is ethyl carboxylate.

PRB-25 corresponds to a compound of formula (I), wherein R ₁ or R ₂ is an ethylamino group and the other is hydrogen, and X is a primary carboxamide.

PRB-26 corresponds to a compound of formula (I), wherein R ₁ or R ₂ is a diethylamino group and the other is hydrogen, and X is ethyl carboxylate.

PRB-2 is the most preferred compound of the present invention. Other preferred compounds of the present invention are PRB-5, PRB-7 and PRB-8.

It should be understood that the present invention includes any possible pharmaceutically acceptable salts, solvates, complexes or prodrugs of the compounds of formula (I).

"Prodrugs" are substances that may or may not have pharmacological activity, as such, but can be introduced into the body (eg, orally or parenterally), where they will undergo bioactivation (for example, metabolized), resulting in an agent of the present invention which is pharmacologically active.

Where X is a carboxylic acid, the present invention also includes salts of a carboxylic acid. Suitable pharmacologically acceptable salts of carboxy groups include metal salts such as, for example, aluminum, alkali metal salts such as lithium, sodium or potassium, alkaline earth metal salts such as calcium, magnesium and ammonium salts, and substituted ammonium salts.

"Therapeutically effective amount" means the amount of a therapeutic agent at which the expected result is achieved. Since the individual needs of the patient may vary, determining the optimal amounts of each nitrite oxide adduct is within the competence of a person skilled in the art. Typically, a regimen for the treatment of diseases and disorders by the compounds and / or compositions of the present invention is selected in accordance with a variety of factors, including the age, weight, gender, diet and general condition of the patient.

By “drug” is meant a compound according to formula (I) in any form suitable for medical use, for example, in the form of a drug product, pharmaceutical product or product, dietary product, food product or food supplement.

In the context of this specification, the term “therapy” also includes “prophylaxis” unless specific indications to the contrary exist. Similarly, the terms “therapeutic” and “therapeutically” are used.

Treatment includes any therapeutic use that may be beneficial to a human or animal. Treatment of mammals is preferred. Both human treatment and animal treatment are within the scope of the present invention. Treatment may be treatment of an existing pathological condition, or it may be prophylactic. Treatment may be in relation to adult subjects, juvenile subjects, children, fetuses, or parts (for example, an organ, tissue, cell or nucleic acid molecule). By "chronic treatment" is meant treatment that lasts for weeks or years.

The term “therapeutically or pharmaceutically active amount” means that amount which results in the desired pharmacological and / or therapeutic effects. The compound of the present invention may, for example, be included in a food product, food supplement, nutritional supplement or dietary product. Alpha-substituted derivatives of DHA and EPA (or DHA for that matter) can be linked together or combined in a triglyceride form by esterification between a mixture of alpha-derivatives, EPA and glycerin catalyzed by Novozym 435 (a commercially available lipase from Candida antarctica in an immobilized form )

The compounds of formula (I) have activity as pharmaceutical products, in particular, as triggers of the activity of nuclear receptors. Thus, the present invention also relates to compounds of formula (I), pharmaceutically acceptable salts, solvates, complexes or prodrugs thereof, as defined above, for use as medicaments and / or for use in therapy. Preferably, the novel compounds or pharmaceutically acceptable salts, solvates, complexes or prodrugs thereof of the present invention can be used:

- for the prevention and / or treatment of diabetes in humans and animals; to control weight loss and / or prevent weight gain;

- for the prevention and / or treatment of obesity or overweight in humans or animals;

- for the treatment and / or prevention of diseases associated with amyloidosis;

- for the treatment or prevention of multiple risk factors for cardiovascular disease;

- for the prevention of cerebrovascular accident, cerebral or transient ischemic attacks associated with atherosclerosis of certain arteries.

- for the treatment of tuberculosis or HIV.

There are two forms of diabetes. One form is type 1 diabetes, which is known as insulin-dependent diabetes mellitus (IDDM), and the other form is type 2 diabetes, which is known as insulin-independent diabetes mellitus (NDDDM). Type 2 diabetes is associated with obesity / overweight and lack of exercise, its onset is often gradual, usually type 2 diabetes occurs in adults and is caused by decreased sensitivity to insulin. This leads to a compensatory increase in insulin production. This stage, before the development of type 2 diabetes itself, is called metabolic syndrome and is characterized by hyperinsulinemia, insulin resistance, obesity, impaired glucose tolerance, increased blood lipids, hypercoagulopathy, dyslipidemia and inflammation, often leading to arteriosclerosis of the arteries. Later, when insulin secretion is impaired, type 2 diabetes develops.

In a preferred embodiment, the compounds of formula (I) can be used to treat type 2 diabetes. The compounds of formula (I) can also be used to treat other types of diabetes selected from the group comprising metabolic syndrome, secondary diabetes, such as pancreatic, extrapancreatic / endocrine, or drug-induced diabetes, or specific forms of diabetes, such as lipoatrophic, myatonic or a disease caused by a violation of insulin receptors. The present invention also includes the treatment of type 2 diabetes. Accordingly, compounds of formula (I), as defined above, can activate nuclear receptors, preferably PPAR (peroxisome proliferation activating receptors) α and / or γ.

The compounds of formula (I) can also be used to treat and / or prevent obesity. Obesity is usually associated with increased insulin resistance, and obese people are at high risk for developing type 2 diabetes, which is a big risk factor for developing cardiovascular disease. Obesity is a chronic disease that affects an increasing number of people in Western countries, obesity is associated not only with moral suffering, but also with reduced life expectancy and many problems, such as diabetes, insulin resistance and high blood pressure. Thus, the present invention meets the long-felt need for a drug that will reduce total body weight or the amount of adipose tissue in obese people, reduce weight to their ideal body weight and without significant unwanted side effects.

Compounds corresponding to formula (I) can also be used for the prevention and / or treatment of diseases associated with amyloidosis. Pathological conditions or diseases associated with amyloidosis are associated with amyloid deposition, preferably as a result of plaque formation, including Alzheimer's disease or dementia, Parkinson's disease, amyotrophic lateral sclerosis, spongiform encephalopathies, Kretzfeld-Jacob, cystic fibrosis, primary and secondary renal fibrosis amyloid deposition in arteries, myocardium and nerve tissue. These diseases can be sporadic, hereditary, or even associated with infections, such as tuberculosis or HIV, and often occur only at a late stage of life, even if non-inherited forms may appear much earlier. Each disease is associated with a particular protein or a combination of these proteins, which are believed to cause pathological conditions associated with the disease. Treatment of diseases associated with amyloidosis can be either short-term (acute) or long-term (chronic).

The compounds of formula (I) can also be used for treatment aimed at reducing amyloid accumulations, preventing incorrect folding of the protein, which can lead to the formation of so-called plaques, treatment aimed at reducing the production of a protein precursor, such as A-protein (beta amyloid protein), and prevention and / or treatment aimed at inhibiting or slowing the formation of accumulations of protein or plaques. Prevention of neurofibrillary tangles by the use of compounds of formula (I) as defined above is also included in the present invention. In one embodiment, new compounds, pharmaceutically acceptable salts, solvates, complexes or prodrugs thereof, as defined above, are used to treat tuberculosis or HIV (human immunodeficiency virus).

Further, the compounds of formula (I) can be used in patients with symptoms of atherosclerosis of the arteries that feed the brain, for example, with cerebrovascular accident or transient ischemic attacks in order to reduce the risk of a future, possibly fatal attack.

The compounds of formula (I) can also be used to treat elevated blood lipids.

In addition, the compounds of formula (I), as defined above, can be used to treat and prevent multiple risk factors for cardiovascular diseases, such as high blood pressure, hypertriglyceridemia, and high coagulation activity of factor VII. Preferably, the compounds of formula (I) are used to treat elevated blood lipids in humans.

The compounds of formula (I) and pharmaceutically acceptable salts, prodrug solvates or complexes thereof can be used on their own, but will usually be administered in the form of a pharmaceutical composition in which the compounds of formula (I) (active ingredient) are bound to a pharmaceutically acceptable excipient, solvent or carrier .

The present invention thus also relates to a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I) of the present invention and a pharmaceutically acceptable carrier, diluent or excipient (including combinations thereof).

This is a pharmaceutical composition that comprises or consists of a therapeutically effective amount of a pharmaceutically active agent. The pharmaceutical composition preferably includes a pharmaceutically acceptable carrier, diluent or excipient (including combinations thereof). Suitable carriers or solvents for therapeutic use are well known in the pharmaceutical field. The choice of pharmaceutical carrier, excipient or solvent depends on the intended route of administration and standard pharmaceutical practice. Pharmaceutical compositions may include (or in addition) a carrier, excipient or solvent, any suitable binder (s), lubricant (s), suspending agent (s), coating agent (s), solubilizing agent (- wa).

Pharmaceutical compositions within the scope of the present invention may include one or more of the following: preservatives, solubilizing agents, stabilizing agents, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, odorants (the salt compounds of the present invention may themselves be in the form of a pharmaceutically acceptable salt) buffers, coating agents, antioxidants, suspending agents, adjuvants, fillers and solvents.

The pharmaceutical composition of the present invention is preferably formulated for oral use in humans and animals. The pharmaceutical composition can also be formulated for any other route of administration, in which the active ingredients can be effectively absorbed and used, for example, intravenously, subcutaneously, intramuscularly, intranasally, rectally, vaginally or topically.

In a specific embodiment of the present invention, the pharmaceutical composition may be in the form of a capsule, or in the form of microcapsules forming a powder, or in the form of a sachet powder. The capsule may be flavored. This embodiment also includes a capsule, wherein both the capsule and the encapsulated fatty acid composition of the present invention are flavored. The flavored capsule is more attractive to the consumer. For the above therapeutic applications, the dosage will, of course, vary depending on the compound used, the route of administration, the treatment desired, and the extent of the body disorders.

The pharmaceutical composition can be formulated so that the daily dosage will be from 10 mg to 10 g. Preferably, the pharmaceutical composition is formulated so that the daily dosage of the above composition is from 5 mg to 5 g. More preferably, the pharmaceutical composition is formulated so that the daily dose the dosage of the above composition is from 100 mg to 1 g. By daily dosage is meant a dosage within 24 hours. The dosage will, of course, vary depending on the compound used, the route of administration, the treatment desired and the degree of disturbance of the body. Typically, the doctor determines the dosage that is most suitable for this subject. The dose and frequency of administration for each individual patient may vary and will depend on many factors, including the activity of the compound used, metabolic stability and duration of action of the compound, age, body weight, general health of the patient, gender, diet, method and time of administration, rate of excretion , a combination of drugs, the severity of the pathological condition and individual therapy. The substance and / or pharmaceutical composition of the present invention can be administered 1 to 10 times a day, for example 1 or 2 times a day. With oral and parenteral administration, the daily dose of the substance can be administered once or can be divided into several doses.

A further aspect of the present invention relates to a fatty acid composition comprising compounds of formula (I). A fatty acid composition comprising compounds of formula (I) enhances the natural biological effects of DHA that result from the regulation of gene expression, and derivatives of the present invention will accumulate in a pool of free fatty acids.

The fatty acid composition may include from 60 to 100% by weight of the compound of formula (I), all percentages by weight based on the total weight of the fatty acid composition. In a preferred embodiment of the present invention, at least 80% by weight of the fatty acid composition is comprised of compounds of formula (I). More preferably, the compounds of formula (I) comprise at least 90% by weight of the fatty acid composition. More preferably, the compounds of formula (I) comprise more than 95% by weight of the fatty acid composition. The fatty acid composition may include at least one of the fatty acids: (full-Z) -5,8,11,14,17-eicosapentaenoic acid (EPA), (full-Z) -4,7,10,13,16 , 19-docosahexaenoic acid (DHA), (all-Z) -6,9,12,15,18-heneukosapentaenoic acid (HPA) and (all-Z) -7,10,13,16,19-docosapentaenoic acid ( DPAn-3), (all-Z) -8,11,14,17-eicosatetraenoic acid (ETAn-3), or combinations thereof. In addition, the fatty acid composition may include (all-Z) -4,7,10,13,16-docosapentaenoic acid (DPAn-6) and / or (all-Z) -5,8,11,14-eicosatetraenoic acid (ARA) or their derivatives. The fatty acid composition may also include these fatty acids or combinations thereof in the form of derivatives. Derivatives are substituted in the same way as the DHA derivatives of formula (I) as defined above.

The fatty acid composition of the present invention may include (full-Z omega-3) -6,9,12,15,18-heneukosapentaenoic acid (HPA) or derivatives thereof in an amount of at least 1% by weight or in an amount of 1 to 4% by weight.

In addition, the fatty acid composition of the present invention may include omega-3 fatty acids, not EPA and not DHA, which have 20, 21 or 22 carbon atoms, or their derivatives in an amount of at least 1.5% by weight or in an amount of at least 3% by weight.

In specific embodiments of the present invention, the fatty acid composition is a pharmaceutical composition, a food composition, or a dietary composition. The fatty acid composition may further include an effective amount of a pharmaceutically acceptable antioxidant. Preferably, the antioxidant is tocopherol or a mixture of tocopherols. In a preferred embodiment, the fatty acid composition further comprises tocopherol or a mixture of tocopherols in an amount of up to 4 mg per 1 g of the total weight of the fatty acid composition. Preferably, the fatty acid composition comprises an amount of tocopherols from 0.2 to 0.4 mg per gram of composition.

Another aspect of the present invention relates to a fatty acid composition, any pharmaceutically acceptable salt, solvate, prodrug or complex thereof, which include compounds of formula (I) as defined above, for use as a medicine and / or in therapy. Such a fatty acid composition can be used to prevent and / or treat the same pathological conditions in which the compounds of formula (I) are used, as described above.

When a fatty acid composition is used as a medicine, it will be administered in a therapeutically or pharmaceutically active amount.

In a preferred embodiment, the composition of fatty acids in humans or animals is administered orally.

The present invention also relates to the use of a compound of formula (I), or a pharmaceutically acceptable salt, solvate, prodrug or complex thereof, as defined above, for the manufacture of a medicament for controlling weight loss and / or preventing weight gain; for the manufacture of a medicament for the treatment and / or prevention of obesity or overweight; for the manufacture of a medicament for the prevention and / or treatment of diabetes in humans or animals; for the manufacture of a medicament for the treatment and / or prevention of diseases associated with amyloidosis; for the manufacture of a medicament for the treatment or prevention of multiple risk factors for cardiovascular diseases such as high blood pressure, hypertriglyceridemia and high coagulation activity of factor VII; for the manufacture of a medicament for the treatment of tuberculosis or HIV; for the manufacture of a medicament for the prevention of cerebrovascular accident, cerebral or transient ischemic attacks associated with atherosclerosis of certain arteries; for the manufacture of a medicament for lowering the level of triglycerides in the blood of mammals and / or increasing the level of high density lipoproteins in patients; or for the manufacture of a medicament for the treatment and / or prevention of a metabolic syndrome. All of these embodiments also include the use of a fatty acid composition as defined above, including components of formula (I) for the manufacture of drugs, as described above. The present invention also relates to the use of alpha-hydroxy-DHA for the manufacture of drugs, as described above.

The present invention also relates to a method for controlling weight loss and / or preventing weight gain, wherein a fatty acid composition comprising at least a compound of formula (I) as defined above is administered to a human or animal.

In addition, the present invention relates to a method for the treatment and / or prevention of obesity or overweight, wherein a fatty acid composition comprising at least a compound of formula (I) as defined above is administered to a human or animal.

In a preferred embodiment of the present invention, the present invention relates to a method for the prevention and / or treatment of diabetes mellitus, wherein a fatty acid composition comprising at least a compound of formula (I) as defined above is administered to a human or animal. Preferably, diabetes mellitus is type 2 diabetes.

Other aspects of the present invention relate to:

- a method for the treatment and / or prevention of diseases associated with amyloidosis;

- a method for the treatment or prevention of multiple risk factors for cardiovascular disease;

- a method for the prevention of cerebrovascular accident, cerebral or transient ischemic attacks associated with atherosclerosis of certain arteries;

wherein a fatty acid composition comprising at least a compound of formula (I), as defined above, is administered to a human or animal.

Derivatives of fatty acids of the formula (I) can be obtained most efficiently based on DHA. If the starting material is not pure DHA (i.e., it is not 100% DHA), the final fatty acid composition will contain a mixture of DHA derivatives as defined above, and some other non-DHA fatty acids, where these fatty acids are replaced by the same way, as new analogues of fatty acids of the formula (I). Such embodiments are also included in the present invention.

In another embodiment of the present invention, compounds of formula (I) are prepared based on (all-Z) -4,7,10,13,16,19-docosahexaenoic acid (DHA), wherein said DHA is derived from a plant source, a microbial source, and / or animal origin or combinations thereof. Preferably, said DHA is derived from fish oil.

The fatty acids in the composition can also be obtained from a source of plant, microbial or animal origin, or a combination thereof. Thus, the present invention also includes a fatty acid composition prepared from microbial fat.

The present invention relates to a method for the preparation of new fatty acid analogues of formula (I) as defined above.

DHA is obtained from biological sources: marine fish fat, microbial fat, vegetable fat. All possible raw materials are mixtures of fatty acids in triglyceride form, where DHA is only a fraction of fatty acids. Typically, the concentration of DHA is 40% in microbial fat and 10-25% in marine fish fat. Vegetable fats containing DHA are under development, and fats with a high concentration of DHA are expected in the future.

The first step in the process will always be the conversion of triglycerides to free fatty acids or monoesters. Preferred esters are ethyl ester or methyl ester. Thus, the fatty acids bound in the triglyceride are separated from one another, and separation is possible. There are several ways to separate DHA from other fatty acids, the most commonly used is a molecular distillation method in which the fatty acids are separated by evaporation, and a separation method by precipitation with urea, in which the fatty acids are separated by degree of unsaturation. The separation of fatty acids is also carried out using the following methods: a method using silver nitrate, in which the fatty acids are separated by degree of unsaturation, esterification reactions catalyzed by lipases, in combination with molecular distillation and countercurrent extraction with carbon dioxide in a supercritical state.

The most important task associated with obtaining pure DHA. is the separation of this fatty acid from other highly unsaturated fatty acids containing 20-22 carbon atoms present in all sources. These fatty acids have properties so similar to DHA that none of the methods listed above provide a sufficient degree of separation. For some microbial fats containing a large amount of DHA and a very low level of highly unsaturated fatty acids containing 20-22 carbon atoms, the application of the molecular distillation method alone or in combination with other methods can provide more than 90% purity.

Fats containing a large amount of DHA also contain significant amounts of highly unsaturated fatty acids containing 20-22 carbon atoms, for example EPA (20: 5n-3), n-3DPA (22: 5n-3), HPA (21: 5n-3 ) and others. The only existing method for separating DHA from such fatty acids is high performance liquid chromatography, silica gel or silica gel impregnated with silver nitrate are used as the stationary phase, and selected organic solvents or supercritical carbon dioxide are used as the mobile phase. Using this method, DHA with more than 97% purity is obtained. However, it should be noted that the technological cost increases significantly with increasing concentration, for example, the technological cost of 97% DHA is more than five times higher than the technological cost of 90% DHA.

DHA, having a purity of 90, 95 or 97%, contains very small amounts of other fatty acids. For example, DHA, having a purity of 97%, contains n-3DPA (22: 5n-3), but also long chain fatty acids, for example EPA (20: 5n-3), HPA (21: 5n-3) and others. However, other fatty acids will react in the same way as DHA and alpha substituted derivatives will be obtained.

Using organic synthesis, a purification method is possible, since DHA and n-6DPA (and 22: 5n-6, which is usually present in very low concentrations) are the only known fatty acids from which gamma-lactones can be obtained by cyclization from the first double communication. The following route can be applied: lactonization, then purification and hydrolysis, but this method is more expensive than HPLC (high performance liquid chromatography).

In one embodiment, the compounds of formula (I), where R ₁ (or R ₂ ) is hydrogen, obtained using the following methods (Scheme 1). Appropriately adapted, these methods can be used to prepare compounds represented by the general formula (I), wherein both R ₁ and R ₂ are, for example, a C ₁ -C ₇ alkyl group, benzyl, halogen, benzyl, alkenyl or alkynyl.

Compounds represented by general formula (I), wherein R ₁ is hydrogen and R ₂ is a C ₁ -C ₇ alkyl group, benzyl, halogen, benzyl, alkenyl, alkynyl, obtained by reacting a DHA ester with a strong non-nucleophilic base, i.e. with substances such as lithium diisopropylamine or potassium / sodium hemethyldisilazidean in a solvent such as tetrahydrofuran, diethyl ether at temperatures from -60 to -78 ° C, to obtain ether enolate (method 1/1 st process).

(Scheme 1)

The ether enolate reacts with an electrophilic reagent similar to an alkyl halide, examples of which are ethyl iodide, benzyl chloride, an acyl halide, examples of which are benzoyl bromide, carboxylic anhydride, an example of which is acetic anhydride, or an electrophilic halogenation reagent, an example of which is N-fluorobenzene , etc. to obtain a monosubstituted derivative (method 2/2 nd process). The ester is further hydrolyzed in a solvent such as ethanol or methanol to a carboxylic acid derivative by adding a base such as lithium / sodium hydroxide in water at temperatures from 15 to 40 ° C.

The Klaisen condensation of DHA ethyl ester occurs during the treatment of DHA ethyl ester with a strong base. This condensation product may have interesting biological activity. Thus, in one embodiment of the present invention, the condensation (intermediate) of the product mentioned above and the use of this product for the treatment and / or prevention of diseases corresponding to the present invention are described.

In another embodiment, the compounds represented by general formula (I) are synthesized using the following methods (Scheme 2).

(Scheme 2)

The compounds represented by general formula (I), wherein R ₁ is hydrogen and R ₂ is hydroxy, alkoxy, acyloxy, are prepared by reacting the DHA ester with a strong non-nucleophilic base, i.e. with substances such as lithium diisopropylamine or potassium / sodium hemethyldisilazidean in a solvent such as tetrahydrofuran, diethyl ether at temperatures from -60 to -78 ° C, to obtain ether enolate (method 4 / process 4). This ether enolate reacts with an oxygen source such as dimethyldioxirane, 2- (phenylsulfonyl) -3-phenyloxaziridine, molecular oxygen with various additives, such as trimethylphosphite or various catalysts like Ni (II) complex to produce alpha-hydroxy-DHA ester ester ( method 5 / process 5). The reaction of a secondary alcohol with a base like sodium hydride in a solvent like THF or DMF gives an alkoxide that reacts with various electrophilic reagents such as alkyl iodide, for example; methyl iodide, ethyl iodide, benzyl bromide or acyl halide, for example; acetyl chloride, benzoyl bromide (method 6 / process 6). The ester is then hydrolysed in a solvent such as ethanol or methanol to a carboxylic acid derivative by adding a base such as lithium / sodium hydroxide in water at temperatures from 15 to 40 ° C. (method 7 / process 7).

The hydroxy-DHA ester is a convenient intermediate for introducing other functional groups into the α-position according to the present invention. The gyroxyl group can be activated by conversion to a halide or tosylate prior to reaction with various nucleophilic compounds such as ammonia, amines, thiols, etc. To convert the hydroxyl group to other functional groups, the Mitsunobu reaction can also be used (Mitsunobu, O, Synthesis, 1981, 1).

Compounds represented by general formula (I) as defined above can also be synthesized by combinations of the various methods described. The present invention includes the above methods.

The present invention also relates to a method for producing a pharmaceutical composition of the present invention, which comprises at least a compound of formula (I) or a pharmaceutically acceptable salt, solvate, complex or prodrug thereof, as defined above, with a pharmaceutically acceptable excipient, solvent or carrier .

Pure enatiomeric compounds can be prepared by cleavage of the racemic compounds of formula (I) as defined above. The cleavage of the compound of formula (I) can be performed using known procedures, for example, by reacting the compound of formula (I), a mixture of diastereoisomers will be obtained, which can be separated by chromatography. After that, the two enantiomers of compound (I) can be obtained from the separated diastereoisomers by, for example, hydrolysis.

Stoichiometric chiral compounds can also be used to effect asymmetric introduction of substituents at the α-position of DHA. The use of chiral oxazolin-2-one proved to be the most effective technique. Enolates obtained from chiral N-acyloxazolidines can be blocked by various electrophiles in a highly stereo-regulated manner (Ager, Prakash, Schaad 1996).

Examples

The present invention will be described in more detail using the following examples, which should not be construed as the only possible in the present invention. In the examples, the structures were checked using mass spectrometry (MS). It should be noted that fatty acid derivatives can also be obtained from starting materials containing low and medium amounts of DHA (i.e., approximately 40-60% by weight of DHA).

Synthesis protocols

Obtaining ethyl ester of α-methyl-DNA (PRB-1)

Butyllithium (228 ml, 0.37 mol, 1.6 M in hexane) was added dropwise to a stirred solution of diisopropylamine (59.5 ml, 0.42 mol) in dry THF (800 ml) in an atmosphere of N ₂ at 0 ° C . The resulting solution was stirred at 0 ° C for 30 minutes, cooled to -78 ° C, and then stirred for an additional 30 minutes until DHA ethyl ester (100 g, 0.28 mol) was added dropwise in dry THF (500 ml) in within 2 hours. The dark green solution was stirred at -78 ° C for 30 minutes before the addition of MeI (28 ml, 0.45 mol). The solution was left for 1.5 hours until it reached a temperature of -20 ° C, then poured into water (1.5 L) and extracted with heptane (2 × 800 ml). The combined organic phases were washed with 1 M Hcl (1 L), dried (Na ₂ SO ₄ ), filtered and evaporated in vacuo. The product was purified by flash chromatography on silica gel, eluting with heptane / EtOAc (99: 1), and 50 g (48%) of the title compound was obtained as a light yellow oil;

¹ H-NMR (200 MHz, CDCl ₃ ) δ 1.02 (t, J 7.5 Hz, 3H), 1.20 (d, J 6.8 Hz, 3H), 1.29 (t, J 7 , 1 Hz, 3H), 2.0-2.6 (m, 5H), 2.8-3.0 (m, 10H), 4.17 (t, J 7.1 Hz, 2H), 5, 3-5.5 (m, 12H);

MC (electrospray ionization); 393 [M + Na].

Obtaining ethyl ester α-ethyl-DHA (PRB-2)

Butyllithium (440 ml, 0.67 mol, 1.6 M in hexane) was added dropwise to a stirred solution of diisopropylamine (111 ml, 0.78 mol) in dry THF (750 ml) in a N ₂ atmosphere at 0 ° C. The resulting solution was stirred at −78 ° C. for 45 minutes until DHA ethyl ether (200 g, 0.56 mol) was added dropwise in dry THF (1.6 L). The addition of ester was completed after 4 hours. The dark green solution was stirred at -78 ° C for 30 minutes before adding EtI (65 ml, 0.81 mol). The solution was left until it reached –40 ° C until an additional amount of EtI was added (5 ml, 0.06 mol), and then the solution was left until it reached –15 ° C (within 3 hours from -78 ° C), after that the mixture was poured into water and extracted with hexane (2x). The combined organic phases were washed with 1 M Hcl, water, dried (Na ₂ SO ₄ ), filtered and evaporated in vacuo. The product was purified by flash chromatography on silica gel, eluting with heptane / EtOAc (99: 1, then 50: 1), and 52.2 g (20%) of the title compound were obtained as a yellow oil.

¹ H-NMR (200 MHz; CDCl ₃ ) δ 0.8-1.0 (m, 6H), 1.2-1.4 (m, 4H), 1.5-1.7 (m, 2H) , 2.12 (m, 2H), 2.3-2.5 (m, 2H), 2.8-3.0 (m, 10H), 4.18 (t, J = 7.1 Hz, 2H ), 5.3-5.6 (m, 12H);

MC (electrospray ionization); 407 [M + Na].

Obtaining ethyl ester α-ethoxy-DNA (PRB-3)

To a suspension of 60% NaH (84.1 mg, 2.1 mmol) in THF, 5 ml, a solution of α-hydroxy-DHA ethyl ester (PRB-12) (372 mg) was added dropwise at −78 ° C. under N ₂ atmosphere. , 1.00 mmol) in THF, 5 ml, the resulting mixture was stirred at -78 ° C for 20 minutes until drops of ethyl iodide (0.24 ml, 3.01 mmol) were added. The reaction mixture was gradually brought to room temperature overnight. A saturated aqueous solution of NH ₄ Cl, 15 ml was added, and the mixture was extracted with diethyl ether, 25 ml × 2, the organic phase was washed with brine, 25 ml, dried (Na ₂ SO ₄ ), filtered and evaporated in vacuo, then flash chromatography on silica gel was performed, elution was carried out using heptane / EtOAc (95: 5), and 68 mg (17%) of the product was obtained as a yellow liquid.

¹ H NMR (200 MHz, CDCl ₃ ) δ 0.94 (t, J = 7.5 Hz, 3H), 1.16-1.29 (m, 6H), 2.05 (quintet, J = 7, 2 Hz, 2H), 2.50 (m, 2H), 2.76-2.84 (m, 10H), 3.33-3.48 (m, 1H), 3.53-3.71 (m , 1H), 3.83 (dd, J = 6.8 Hz, J = 6.2 Hz, 1H), 4.18 (q, J = 7.1 Hz, 2H), 5.31-5.45 (m, 12H)

¹³ C NMR (50 MHz, CDCl ₃ ) δ 14.2, 15.1, 20.5, 25.5, 25.6, 25.7, 31.0, 60.8, 66.0, 78.7 , 124.1, 127.0, 127.8, 127.9, 128.0 (2 signals), 128.2 (2 signals), 128.5, 130.7, 132.0, 172.5 (3 hidden signal)

MC (electrospray ionization); 423 [M + Na] ⁺

Obtaining ethyl ester of α-fluoro-DHA (PRB-4)

To LDA (2.1 ml, 4.2 mol 2 M in THF / heptane / ethylbenzene) in dry THF (10 ml) in an atmosphere of N ₂ at -78 ° C was added dropwise over 15 minutes DHA ethyl ester (1 g, 2.8 mmol) in dry THF (30 ml). Then NFSi (1.06 g, 3.4 mmol) was added. The solution was left until it reached room temperature and then stirred for 70 hours. The mixture was poured into water and extracted with hexane (2x). The combined organic phases were washed with 1 M HCl, water, dried (Na ₂ SO ₄ ), filtered and evaporated in vacuo; MS (electrospray ionization); 397 [M + Na].

Obtaining ethyl ester of α, α-dimethyl DHA (PRB-5)

Butyllithium (100 ml, 0.17 mol, 1.6 M in hexane) was added dropwise to a stirred solution of diisopropylamine (28 ml, 0.20 mol) in dry THF (100 ml) in an atmosphere of N ₂ at 0 ° C. The resulting solution was stirred at 0 ° C for 30 minutes, cooled to -78 ° C and a solution of DHA ethyl ester (50 g, 0.14 mol) in dry THF (200 ml) was added dropwise. The resulting dark green solution was stirred at -78 ° C for 30 minutes before the addition of MeI (17 ml, 0.28 mol). The solution was left until it reached -10 ° C, then poured into water and extracted with heptane (2x). The combined organic phases were washed with 1 M Hcl (1 L), dried (Na ₂ SO ₄ ), filtered and evaporated in vacuo.

The procedure was repeated, but the crude α-methyl-DHA ethyl ester product was used in place of DHA ethyl ester. The product was purified by flash chromatography on silica gel, heptane / EtOAc (99: 1, then 98: 2), and 31.6 g (59%) of the title compound were obtained as a pale yellow oil;

¹ H-NMR (200 MHz, CDCl ₃ ) δ 1.01 (t, J 7.5 Hz, 3H), 1.21 (s, 6H), 1.28 (t, J 7.1 Hz, 3H) 2.08 (m, 2H), 2.34 (d, J 6.8 Hz, 2H), 2.8-3.0 (m, 10H), 4.15 (q, J 7.5 Hz, 2H), 5.3-5.6 (m, 12H);

¹³ C-NMR (50 MHz, CDCl ₃ ) δ 14.7, 21.0, 25.3, 26.0, 26.1, 38.3, 42.8, 60.7, 125.8, 127, 4, 128.3, 128.5, 128.6, 128.7, 129.0, 130.7, 132.4, 177.9;

MS (electrospray ionization); 385 [M + H].

Obtaining ethyl ester of α-thiomethyl-DNA (PRB-6)

Α-iodo-DHA ethyl ester (0.5 g, 1.04 mmol) was dissolved in 20 ml of THF in an atmosphere of N ₂ at 0 ° C. MeSNa (80 mg, 1.14 mmol) was added and the mixture was stirred for several minutes before it was diluted with heptane. The organic phase was washed with water (2x), dried (Na ₂ SO ₄ ) and evaporated in vacuo. The desired product was purified by flash chromatography, eluting with heptane / EtOAc (30: 1), and α-thiomethyl-DHA ethyl ester was obtained as a pale yellow oil. Α-thiomethyl-DHA ethyl ester was dissolved in 10 ml EtOH and 10 ml THF. LiOH (0.39 g, 9.2 mmol) dissolved in 5 ml of water was added to the solution. The reaction mixture was stirred overnight at room temperature, then the mixture was diluted with water and heptane.

The organic phase was extracted with 1 M LiOH (2x) and the combined aqueous phases were acidified with 5 M Hcl and extracted with diethyl ether (2x). The combined organic phases were washed with brine, water, dried (Na ₂ SO ₄ ) and evaporated in vacuo to give 183 mg (47%) of the title compound as a pale yellow oil;

¹ H-NMR (200 MHz, CDCl ₃ ) δ 0.98 (t, J 6.6 Hz, 3H), 1.95-2.65 (m, 7H), 2.72-3.05 (m, 10H), 3.12-3.43 (m, 1H), 5.20-5.70 (m, 12H), 10.65 (br s, 1H);

¹³ H-NMR (50 MHz, CDCl ₃ ) δ 14.7, 21.0, 25.9, 26.0, 26.2, 28.8, 125.4, 127.4, 128.1, 128, 3, 128.4, 128.7, 128.9, 129.0, 131.6, 132.4, 177.0.

Obtaining ethyl ester of α-thioethyl-DHA (PRB-7)

Α-iodo-DHA ethyl ester (11 g, 23 mmol) was dissolved in 100 ml of THF in an atmosphere of N ₂ at 0 ° C.

EtSNa (2.1 g, 25 mmol) was added and the mixture was stirred for 1 hour at 0 ° C. The reaction was stopped using 1 M Hcl and dilution was carried out using heptane. The organic phase was washed with water (2x), dried (Na ₂ SO ₄ ) and evaporated in vacuo. The desired product was purified by flash chromatography, heptane / EtOAc (30: 1), and 7.3 g (76%) of the title compound were obtained as a pale yellow oil;

¹ H-NMR (200 MHz, CDCl ₃ ) δ 1.1-1.3 (m, 9H), 2.05 (m, 2H), 2.3-2.7 (m, 4H), 2.7 -2.9 (m, 10H), 3.25 (m, 1H), 4.17 (q, J 7.1 Hz, 2H), 5.3-5.5 (m, 12H);

MS (electrospray ionization): 439 [M + Na].

Obtaining ethyl ester of α, α-diethyl-DNA (PRB-8):

Butyllithium (38.6 ml, 0.62 mol, 1.6 M in hexane) was added dropwise to a stirred solution of diisopropylamine (9.1 ml, 0.65 mol) in dry THF (200 ml) under N ₂ at 0 ° C. The resulting solution was stirred at 0 ° C for 30 minutes, cooled to -78 ° C and a solution of DHA ethyl ether (20.0 g, 0.56 mol) in dry THF (100 ml) was added dropwise. The resulting dark green solution was stirred at -78 ° C for 30 minutes before adding EtI (6.8 ml, 0.84 mol). The solution was left until it reached -10 ° C, then poured into water and extracted with hexane (2x). The combined organic phases were washed with 1 M Hcl, dried (Na ₂ SO ₄ ), filtered and evaporated in vacuo.

The procedure was repeated, but the crude α-ethyl-DHA ethyl ester product was used in place of DHA ethyl ester. After addition of EtI, the reaction mixture was allowed to reach room temperature and was stirred overnight. The product was purified by flash chromatography on silica gel, eluting with heptane / EtOAc (99: 1, then 98: 2), and 10.0 g (43%) of the title compound was obtained as a light yellow oil;

¹ H-NMR (200 MHz, CDCl ₃ ) δ 0.83 (t, J 7.4 Hz, 6H), 0.94 (t, J 5.8 Hz, 3H), 1.28 (t, J 7 , 1 Hz, 3H), 1.63 (q, 77.4 Hz, 4H), 2.10 (m, 2H), 2.34 (d, J 6.9 Hz, 2H), 2.8-3 0 (m, 10H), 4.15 (q, J 7.5 Hz, 2H), 5.3-5.6 (m, 12H);

¹³ C-NMR (50 MHz, CDCl ₃ ) δ 8.9, 14.7, 21.0, 23.1, 25.9, 26.0, 26.2, 27.4, 31.2, 50.1, 60.6, 125.5, 127.4, 128.3, 128.6, 128.9, 130.5, 132.4, 177.1;

MS (electrospray ionization); 413.3 [M + H], 435.3 [M + Na].

Obtaining ethyl ester of α-benzyl-DNA (PRB-9)

To a stirred solution of diisopropylamine (0.91 ml, 6.46 mmol) in dry THF (20 ml) in an inert atmosphere at 0 ° C was added drops of n-BuLi (1.6 M in hexane, 3.86 ml, 6, 18 mmol). The mixture was stirred at 0 ° C for 30 minutes, brought to -78 ° C and then stirred at this temperature for 5 minutes. DHA ethyl ether (2.0 g, 5.62 mmol) in dry THF (10 ml) was added dropwise and the mixture was stirred at -78 ° C for 20 minutes, then benzyl bromide (0.80 ml, 6) was added. , 74 mmol). The resulting solution was kept for 3 hours until its temperature became equal to 0 ° C, then it was subjected to distribution between water (100 ml) and heptane (100 ml). The aqueous layer was extracted with heptane and the combined organic layers were washed with 1 M HCl and dried (Na ₂ SO ₄ ). Concentration was carried out under reduced pressure and purification by flash chromatography (Heptane: EtOAc 99: 1), and 1.05 g (42%) of the title compound was obtained as a colorless oil;

¹ H-NMR (200 MHz, CDCl ₃ ): δ 0.99 (t, 3H), 1.18 (t, 3H), 2.08-2.16 (m, 2H), 2.35-2, 42 (m, 2H), 2.74-2.98 (m, 13H), 4.09 (q, 4H), 5.38-5.50 (m, 10H), 7.19-7.36 ( m, 5H);

¹³ C-NMR (50 MHz, CDCl ₃ ): δ 14.61, 14.71, 20.99, 25.98, 26.07, 30.07, 38.32, 48.02, 60.88, 126 , 75, 126.83, 127.46, 128.31, 128.45, 128.53, 128.58, 128.86, 128.77, 129.01, 129.35, 130.55, 132.46 , 138.89, 175.39.

MS (electrospray ionization): 447.3 [M + H], 469.3 [M + Na].

Obtaining ethyl ester α-ethanesulfenyl-DNA (PRB-10)

To a solution of α-thioethyl-DHA ethyl ester (0.5 g, 1.3 mmol) in 15 ml of CHCl ₃ in an atmosphere of N ₂ at -20 ° C was added a solution of MCPBA (0.22 g, 1.3 mmol) in 10 ml CHCl ₃ . The reaction mixture was stirred for 2 hours at this temperature, then the mixture was filtered and washed with saturated aqueous NaHCO ₃ . The aqueous phase was extracted twice with CHCl ₃ and the combined organic phase was washed with water and brine, dried over Na ₂ SO ₄ , filtered and concentrated. The product was purified by flash chromatography using hexane: EtOAc 8: 2, and 0.35 g (70%) of the title compound was obtained.

¹ H NMR (200 MHz, CDCl ₃ ): δ 0.99 (t, 3H), 1.27-1.45 (m, 6H), 2.09 (m, 2H), 2.79-2.94 (m, 14H), 3.55 (m, 1H), 4.25 (q, 2H), 5.37-5.59 (m, 12H).

¹³ C-NMR (50 MHz, CDCl ₃ ): δ 7.97, 14.58, 14.68, 20.95, 23.68, 25.17, 25.93, 26.04, 44.20, 45.15 , 62.30, 64.08, 123.91, 124.47, 127.41, 127.86, 128.26, 128.40, 128.44, 128.72, 128.72, 128.96, 129 12, 132.42, 132.47, 174.55.

MS (electrospray ionization): 455.3 [M + Na].

Obtaining ethyl ester of α-thiophenyl-DNA (PRB-11)

To a solution of α-iodo-DHA ethyl ester (PRB-15) (3.40 g, 7.05 mmol) in acetone (20 ml) was added dropwise a solution of sodium phenyl sulfide (1.039 g, 7.86 mmol) in acetone (110 ml) . The resulting mixture was stirred at room temperature for half an hour, the mixture was evaporated in vacuo, then flash chromatography was carried out on silica gel, elution was carried out using heptane / EtOAc 200: 1-95: 5, and as a result 2.35 g (72 %) of the product in the form of a yellow liquid.

¹ H NMR (200 MHz, CDCl ₃ ): δ 0.97 (t, J = 7.5 Hz, 3H), 1.18 (t, J = 7.1 Hz, 3H), 2.09 (quintet, J = 7, l Hz, 2H), 2.54-2.66 (m, 2H), 2.83-2.86 (m, 10 N), 3.67 (dd, J = 6.8 Hz, J = 8.3 Hz, 1H), 4.12 (quintet, J = 7.1 Hz, 2H), 5.24-5.49 (m, 12H), 7.28-7.33 (m, 3H ), 7.46-7.50 (m, 2H)

¹³ C NMR (50 MHz, CDCl ₃ ): δ 14.0, 14.2, 20.5, 25.5, 25.6. 25.7, 29.4, 50.6, 61.1, 125.1, 127.0, 127.7, 127.9, 128.0, 128.3, 128.42, 128.45, 128, 9, 131.2, 132.0, 133.0, 133.2, 174.1 (5 hidden signals)

MS (electrospray ionization); 465 [M + H] ⁺ , 487 [M + Na] ⁺

High Resolution Mass Spectroscopy (HRMS) (EI (Electron Impact Ionization)) calculated for C ₃₀ H ₄₀ O ₂ S: 464.2749, found: 464.2741

Obtaining ethyl ester of α-hydroxy-DNA (PRB-12)

To a solution of diisopropylamine (19.76 ml, 140 mmol) in dry THF, 40 ml, in an atmosphere of N ₂ at -78 ° C was added drops of 1.6 M BuLi in hexane (87.5 ml, 140 mmol). The resulting mixture was stirred at -78 ° C for 15 minutes before a solution of DHA ethyl ester (24.99 g, 70.1 mmol) in THF (80 ml) was added dropwise. The resulting dark green reaction mixture was stirred for 1 hour at -78 ° C before triethyl phosphite (12.2 ml, 70.1 mmol) was added dropwise and then O _{2 was} passed through the reaction mixture overnight, the reaction mixture kept at -78 ° C for 5 hours and then slowly warmed to room temperature. A saturated aqueous solution of NaHCO ₃ (100 ml) was added and the mixture was extracted with diethyl ether, 200 ml × 2. The organic phase was dried (Na ₂ SO ₄ ), filtered and evaporated in vacuo, then flash chromatography on silica gel was carried out, eluting was carried out using heptane / EtOAc 99: 1-95: 5, and as a result, 4.52 g (17%) of the product was obtained as a yellow liquid.

¹ H NMR (200 MHz, CDCl ₃ ): δ 0.92 (t, J = 7.5 Hz, 3H), 1.24 (t, J = 7.1 Hz, 3H), 2.02 (quintet, J = 7.1 Hz, 2H), 2.44-2.54 (m, 2H), 2.74-2.87 (m, 10 H), 4.13-4.24 (m, 3H), 5.25-5.94 (m, 12H)

¹³ C-NMR (50 MHz, CDCl ₃ ): δ 14.0, 14.1, 20.4, 25.4, 25.5, 25.6, 32.0, 61.5, 69.9, 123 , 3, 126.9, 127.7, 127.9, 128.08, 128.1, 128.2, 128.4, 131.3, 131.8, 174.4 (4 hidden signals)

MS (electrospray ionization); 395 [MH-Na] ⁺

HRMS (ES) calculated for C ₂₄ H ₃₆ O ₃ Na: 395.2556, found: 395.2543

Obtaining amide α-methyl-DNA (PRB-13)

To a solution of α-methyl-DHA (PRB-1 FA) (3.13 g, 9.1 mmol) and oxalyl chloride (8.0 ml, 94.5 mmol) in toluene (90 ml) was added DMF (0.1 ml) and the resulting mixture was stirred at room temperature in an atmosphere of N ₂ for 15 ½ hours. The mixture was then evaporated in vacuo and the precipitate was dissolved in THF, 100 ml, the solution was cooled to 0 ° C, then aqueous NH ₃ (20 ml) was added dropwise. The ice bath was removed and the mixture was stirred at room temperature for 4 hours, then 50 ml of water was added and the aqueous phase was extracted with diethyl ether, 2 × 100 ml. The organic phase was washed with a saturated aqueous solution of NH ₄ Cl, 50 ml, dried (Na ₂ SO ₄ ), filtered and evaporated in vacuo, then flash chromatography on silica gel was carried out, elution was carried out using CH ₂ Cl ₂ / 2M NH ₃ in MeOH 97.5: 2.5, and as a result, 2.51 g (80%) of the product was obtained as a yellow liquid.

¹ H NMR (200 MHz, CDCl ₃ ) δ 0.91 (t, J = 7.5 Hz, 3H), 1.10 (d, J = 9.8 Hz, 3H), 1.94-2.11 (m, 3H), 2.19-2.35 (m, 2H), 2.76-2.77 (m, 10 H), 5.18-5.45 (m, 12 H), 6.03 (s, 1H), 6.72 (s, 1H)

¹³ C NMR (50 MHz, CDCl ₃ ) δ 14.6, 17.6, 20.8, 25.8, 25.9, 32.0, 41.0, 127.3, 128.1, 128.4 , 128.6, 128.8, 130.1, 132.2, 179.6 (8 hidden signals)

MS (electrospray ionization); 342 [M + H] ⁺ , 364 [M + Na] ⁺

HRMS (EI) calculated for C ₂₃ H ₃₅ NO: 341.2719, found: 341.2707

Obtaining ethyl ester α-methoxy-DHA (PRB-14)

To a suspension of 60% NaH (61.1 mg, 1.53 mmol) in THF (5 ml) in an atmosphere of N ₂ at -78 ° C, a solution of α-hydroxy-DHA ethyl ester (PRB-12) (373 mg) was added dropwise 1.00 mmol) in THF (5 ml), the resulting mixture was stirred at -78 ° C for 20 minutes before methyl iodide (0.13 ml, 2.09 mmol) was added dropwise. The reaction mixture was gradually brought to room temperature over 5 hours. A saturated aqueous solution of NH ₄ Cl, 15 ml, was added, and the mixture was extracted with diethyl ether (25 ml × 2), the organic phase was washed with brine (25 ml), dried (Na ₂ SO ₄ ), filtered and evaporated in vacuum, then flash chromatography was carried out on silica gel, elution was carried out with heptane / EtOAc (99: 1-4: 1), and 136 mg (35%) of the product was obtained as a yellow liquid.

¹ H NMR (200 MHz, CDCl ₃ ) δ 0.92 (t, J = 7.5 Hz, 3H), 1.24 (t, J = 7.1 Hz, 3H), 2.03 (quintet, J = 7.3 Hz, 2H), 2.48 (t, J = 5.7 Hz, 2H), 2.73-2.82 (m, 10 H), 3.34 (s, 3H), 3, 74 (t, J = 6.2 Hz, IH), 4.17 (q, J = 7.1 Hz, 2 H), 5.24-5.43 (m, 12H)

¹³ C NMR (50 MHz, CDCl ₃ ) δ 14.1, 20.4, 25.4, 25.5, 25.7, 30.6, 57.9, 60.9, 80.8, 123.7 , 126.9, 127.71, 127.73, 127.92, 127.94, 128.07, 128.1, 128.2, 128.4, 130.7, 131.8, 171.9 (3 hidden signal)

MS (electrospray ionization); 409 [M + Na] ⁺

HRMS (ES) calculated for C ₂₅ H ₃₈ O ₃ Na: 409.2713, found: 409.2711

Obtaining ethyl ester of α-iodo-DHA (PRB-15)

Diisopropylamine (20 ml, 140 mmol) was dissolved in 150 ml of THF in an atmosphere of N ₂ at -20 ° C. BuLi (88 ml, 140 mmol, 1.6 M) was added dropwise to the mixture before the solution was cooled to -78 ° C. DHA ethyl ether (50 g, 140 mmol) in 250 ml of THF was added dropwise to the solution, and the reaction mixture was stirred for 30 minutes at room temperature. The resulting mixture was added dropwise to a solution of I ₂ (42.8 g, 169 mmol) in 400 ml of THF in an atmosphere of N ₂ at -70 ° C. The reaction was stopped using 1 M Hcl and dilution was carried out using heptane. The organic phase was washed with 10% Na ₂ S ₂ O ₃ (2x), dried (Na ₂ SO ₄ ), filtered and evaporated in vacuo. The desired product was purified by flash chromatography, Heptane / EtOAc (100: 1) was used as eluent, and 11.0 g (16%) of the title compound was obtained as a pale yellow oil;

MS (Electrospray Ionization): 505 [M + Na].

Obtaining ethyl ester of α-iodo-DNA (PRB-15)

To a solution of diisopropylamine (42 ml, 298 mmol) in dry THF (150 ml) in an atmosphere of N ₂ at -78 ° C, 1.6 M BuLi in hexane (158 ml, 253 mmol) was added dropwise. The resulting mixture was stirred at -78 ° C for 35 minutes, then a solution of DHA ethyl ester (75.05 g, 210 mmol) in THF (300 ml) was added dropwise. The resulting dark green reaction mixture was stirred for 30 minutes at -78 ° C before adding dropwise a solution of I ₂ (91.06 g, 359 mmol) in THF (200 ml). The reaction mixture was stirred at -78 ° C for 20 minutes, then the reaction was stopped with water (200 ml), extraction was carried out with heptane (300 ml). The organic phase was washed with 1 M Hcl (150 ml) and water (200 ml), dried (Na ₂ SO ₄ ), filtered and evaporated in vacuo. The crude product was purified by flash chromatography on silica gel, heptane / EtOAc (100: 1) was used as eluent, and 26.14 g (26%) of the product was obtained as a yellow liquid.

¹ H NMR (200 MHz, CDCl ₃ ) δ 0.94 (t, J = 7.5 Hz, 3H), 1.24 (t, J = 7.1 Hz, 3H), 2.04 (quintet, J = 7.1 Hz, 2H), 2.69-2.84 (m, 12 H), 4.17 (q, J = 7, l Hz, 2H), 4.22 (t, J = 7.9 Hz, 1H), 5.24-5.49 (m, 12 H)

¹³ C NMR (50 MHz, CDCl ₃ ) δ 13.7, 14.2, 25.5, 26.0 (2 signals), 25.8, 34.0, 61.7, 126.1, 127.0 , 127.4, 127.8, 127.9, 128.0, 128.2, 128.5, 128.5, 131.6, 131.9, 170.9 (4 hidden signals)

MS (electrospray ionization); 505 [M + Na] ⁺

Obtaining ethyl ester of α-amino-DNA (PRB-17)

To a solution of α-phthalimide-DHA ethyl ester (313.5 mg, 0.62 mmol) in ethanol (5 ml) was added hydrazine hydrate (46 μl, 0.95 mmol) and the resulting mixture was refluxed under N ₂ atmosphere for 15 ½ hours, followed by evaporation in vacuo, purification was carried out using flash chromatography on silica gel, elution was carried out using CH ₂ Cl ₂ : 7M NH ₃ in methanol (99: 1-95: 1), and 149 mg was obtained (64%) of the product as a yellow liquid.

¹ H NMR (200 MHz, CDCl ₃ ) δ 0.91 (t, J = 7.5 Hz, 3H), 1.22 (t, J = 7.1 Hz, 3H), 1.72 (bs, 2H ), 2.02 (quintet, J = 7.2 Hz, 2H), 2.39-2.46 (m, 2H), 2.73-2.82 (m, 10 H), 3.47 (bs , 1H), 4.13 (q, 2H), 5.23-5.56 (m, 12 H)

¹³ C NMR (50 MHz, CDCl ₃ ) δ 14.1, 20.4, 25.4, 25.5, 25.6, 54.1, 60.8, 124.4, 126.9, 127.7 (2 signals), 127.9, 128.2, 128.3, 128.4, 131.4, 131.9, 189.3 (6 hidden signals)

MS (electrospray ionization); 372 [M + H] ⁺

Obtaining ethyl ester (S) - (+) - α-ethyl DHA (PRB-20)

Synthesis of Intermediate PRB-18:

DHA (3.00 g, 18.3 mmol) was dissolved in dry CH ₂ Cl ₂ (120 ml) at 0 ° C in an inert atmosphere and DMAP (2.45 g, 20.1 mmol) and DCC (3 96 g, 19.2 mmol). The mixture was stirred at 0 ° C for 20 minutes, then (4R, 5S) - (+) - 4-methyl-5-phenyl-2-oxazolidinone (3.24 g, 18.3 mmol) was added and the resulting mixture was stirred at room temperature for 20 hours. The product was filtered and purification by flash chromatography (heptane: ethyl acetate 6: 1) gave 3.00 g (34%) of intermediate PRB-18 as a colorless oil.

¹ H-NMR (200 MHz, CDCl ₃ ): δ 0.93-1.05 (t + d, 6H), 2.11 (m, 2H), 2.51 (m, 2H), 2.80- 3.00 (m, 10H), 3.05 (m, 2H), 4.77 (m, 1H), 5.34-5.68 (m, 12H), 5.70 (d, 1H), 7 28-7.32 (m, 2H); 131-1 Al (m, 3H).

Synthesis of Intermediate PRB-19

Compound PRB-18 (1.80 g, 3.70 mmol) in dry THF (10 ml) was added dropwise to a solution of LiHMDS (1M in THF, 4.00 ml, 4.00 mmol) in dry THF (15 ml) at -78 ° C in an inert atmosphere. The mixture was stirred at −78 ° C. for 30 minutes, EtI (0.89 ml, 11.1 mmol) was added, and the mixture was slowly brought to 0 ° C. over the course of an hour. The mixture was then stirred at 0 ° C for 18 hours and partitioned between saturated NH ₄ C1 (50 ml) and diethyl ether (50 ml). The aqueous layer was extracted with diethyl ether (50 ml), the combined organic layer was washed with 0.1 M HCl (50 ml) and brine (50 ml). Drying was carried out over Na ₂ SO ₄ , purification by flash chromatography (heptane: ethyl acetate 95: 5) gave a yield of 0.52 g (27%) of intermediate PRB-19 as a colorless oil.

¹ H-NMR (200 MHz, CDCl ₃ ): δ 0.88-1.01 (m, 9H), 1.64-1.78 (m, 2H), 2.08 (m. 2H), 2, 31 (m, 1H), 2.48 (m, 1H), 2.87 (m, 10H), 3.87 (m, 1H), 4.75 (m, 1H), 5.32 (m, 12H ), 5.63 (d, .77.1 Hz, 1H), 7.32 (m, 2H), 7.42 (m, 3H).

¹³ C-NMR (50 MHz, CDCl ₃ ): δ 7.26, 11.75, 14.67, 14.98, 20.95, 25.57, 25.93, 26.04, 29.93, 44 59, 55.31, 79.10, 125.21, 126.01, 127.17, 127.42, 128.27, 128.50, 128.55, 128.67, 128.95, 129.09 130.35, 132.42, 133.80, 153.18, 176.25.

MS (electrospray ionization): 538.2 [M + Na] ⁺

Compound PRB-19 (0.25 g, 0.485 mmol) was dissolved in absolute ethanol (5 ml) and the temperature was brought to 0 ° C in an inert atmosphere. NaOEt (1 M in ethanol, 0.54 ml, 0.54 mmol) was added and the mixture was stirred at 0 ° C for 30 minutes and partitioned between water and heptane. The aqueous layer was extracted with heptane and the combined organic layer was washed with 0.1 M HCl and dried. Purification by flash chromatography afforded 0.025 g (13%) of the title compound PRB-20 as a colorless oil.

¹ H-NMR (200 MHz, CDCl ₃ ) δ 0.8-1.0 (m, 6H), 1.2-1.4 (m, 4H), 1.5-1.7 (m, 2H) , 2.12 (m, 2H), 2.3-2.5 (m, 2H), 2.8-3.0 (m, 10H), 4.18 (t, 2H), 5.3-5 6 (m, 12H).

MS (electrospray ionization); 407 [M + Na].

[α] D + 1.7 ° (c = 1.5, ethanol).

Obtaining ethyl ester (R) - (-) - α-ethyl-DNA (PRB-21):

Synthesis of Intermediate PRB-21:

DHA (1.00 g, 3.05 mmol) was dissolved in dry CH ₂ Cl ₂ (20 ml) at 0 ° C in an inert atmosphere and DMAP (0.41 g, 3.35 mmol) and DCC (0 66 g, 3.20 mmol). The mixture was stirred at 0 ° C for 20 minutes, (4S, 5R) - (-) - 4-methyl-5-phenyl-2-oxazolidinone (0.54 g, 3.05 mmol) was added and the mixture was stirred at room temperature for 20 hours. Filtration and purification by flash chromatography (heptane: ethyl acetate 6: 1) gave 1.08 g (73%) of intermediate PRB-21 as a colorless oil.

¹ H-NMR (200 MHz, CDCl ₃ ): δ 0.93-1.05 (t + d, 6H), 2.11 (m, 2H), 2.51 (m, 2H), 2.80- 3.00 (m, 10H), 3.05 (m, 2H), 4.77 (m, 1H), 5.34-5.68 (m, 12H), 5.70 (d, 1H), 7.28.7 32 (m, 2H); 7.37-7.47 (m, 3H).

Synthesis of Intermediate PRB-22:

Compound PRB-21 (3.25 g, 6.67 mmol) in dry THF (15 ml) was added dropwise to a solution of LiHMDS (1M in THF, 7.34 ml, 7.34 mmol) in dry THF (35 ml) at -78 ° C in an inert atmosphere. The mixture was stirred at -78 ° C for 30 minutes, EtI (1.6 ml, 20.0 mmol) was added and the mixture was slowly brought to 0 ° C over an hour. The mixture was then stirred at 0 ° C for 18 hours and partitioned between saturated NH ₄ Cl (50 ml) and diethyl ether (50 ml). The aqueous layer was extracted with diethyl ether (50 ml), the combined organic layer was washed with 0.1 M HCl (50 ml) and brine (50 ml). Drying was carried out over Na ₂ SO ₄ , purification by flash chromatography (heptane: ethyl acetate 95: 5) gave 1.50 g (44%) of intermediate PRB-22 as a colorless oil.

¹ H-NMR (200 MHz, CDCl ₃ ): δ 0.88-1.01 (m, 9H), 1.64-1.78 (m, 2H), 2.08 (m, 2H), 2, 31 (m, 1H), 2.48 (m, 1H), 2.87 (m, 10H), 3.87 (m, 1H), 4.75 (m, 1H), 5.32 (m, 12H ), 5.63 (d, J7, l Hz, 1H), 7.32 (m, 2H), 7.42 (m, 3H).

¹³ C-NMR (50 MHz, CDCl ₃ ): δ 7.26, 11.75, 14.67, 14.98, 20.95, 25.57, 25.93, 26.04, 29.93, 44 59, 55.31, 79.10, 125.21, 126.01, 127.17, 127.42, 128.27, 128.50, 128.55, 128.67, 128.95, 129.09 130.35, 132.42, 133.80, 153.18,176.25.

MS (electrospray ionization): 538.2 [M + Na]

Compound PRB-22 (0.25 g, 0.485 mmol) was dissolved in absolute EtOH (5 ml) and the temperature was brought to 0 ° C in an inert atmosphere. NaOEt (1M in ethanol, 0.54 ml, 0.54 mmol) was added and the mixture was stirred at 0 ° C for 30 minutes and the mixture was partitioned between water and heptane. The aqueous layer was extracted with heptane and the combined organic layer was washed with 0.1 M HCl and dried. Purification by flash chromatography afforded (13%) the title compound PRB-23 as a colorless oil.

¹ H-NMR (200 MHz; CDCl ₃ ) δ 0.8-1.0 (m, 6H), 1.2-1.4 (m, 4H), 1.5-1.7 (m, 2H) , 2.12 (m, 2H), 2.3-2.5 (m, 2H), 2.8-3.0 (m, 10H), 4.18 (t, 2H), 5.3-5 6 (m, 12H);

MS (electrospray ionization); 407 [M + Na].

[α] D-1.3 ° (c = 1.00, ethanol).

Obtaining ethyl ester of α-phthalimide-DNA (PRB-24)

A mixture of α-hydroxy-DHA ethyl ester (PRB-12) (373.5 mg, 1.00 mmol), phthalimide (178 mg, 1.21 mmol) and triphenylphosphine (313.9 mg, 1.20 mmol) in THF (10 ml), was cooled to 0 ° C in an atmosphere of N ₂ , then diisopropylazodicarboxylate (0.24 ml, 1.24 mmol) was added dropwise. The ice bath was removed and the reaction mixture was stirred at room temperature for 18 hours, after which it was evaporated in vacuo and flash chromatography was carried out on silica gel using heptane / ethyl acetate (99: 1-95 as eluent) : 1), the result was 323 mg (64%) of the product as a yellow liquid.

¹ H NMR (200 MHz, CDCl ₃ ) δ 0.95 (t, J = 7.5 Hz, 3H), 1.22 (t, J = 7.1 Hz, 3H), 2.05 (m, 2H ), 2.72-2.84 (m, 11 H), 3.02-3.22 (1H), 4.20 (q, 7 = 7.1 Hz, 2H), 4.87 (dd, J = 11 Hz, J = 4.9 Hz, 1H), 5.17-5.40 (m, 12H), 7.68-7.75 (m, 2H), 7.79-7.85 (m, 2H)

¹³ C NMR (50 MHz, CDCl ₃ ) δ 14.0, 14.1, 20.4, 25.4, 25.4, 25.5, 27.0, 51.8, 61.7, 123.8 , 124.3, 126.9, 127.5, 127.7, 127.9, 127.9, 128.1, 128.1, 128.3, 128.4, 131.6, 131.8, 131 , 8, 134.0, 167.3, 168.7 (2 hidden signals)

MS (electrospray ionization); 502 [M + H] ⁺ , 524 [M + Na] ⁺

Preparation of α-ethylamino-DHA ethyl ester (PRB-25) and α-diethylamino-DHA ethyl ester (PRB-26)

To a mixture of α-amino-DHA ethyl ester (PRB-17) (746.5 mg, 2.01 mmol), LiOH · H ₂ O (171.6 mg, 4.09 mmol) and molsieve 4A (599 mg) in Ethyl bromide (3.0 ml, 40.2 mmol) was added DMF (4 ml), and the resulting mixture was stirred at room temperature for 71 hours. The mixture was diluted with diethyl ether (100 ml) and filtered. The organic phase was washed with 1 M NaOH (20 ml) and brine (20 ml), dried (Na ₂ SO ₄ ), filtered and evaporated in vacuo, purification was carried out by flash chromatography on silica gel, elution was carried out using heptane: ethyl acetate (95: 5) —CH ₂ Cl ₂ : 2M NH ₃ in methanol (99: 1), resulting in 458 mg (53%) of PRB-26 as a yellow liquid and 152 mg (19%) of PRB-25 in in the form of a yellow liquid.

PRB-26:

¹ H NMR (200 MHz, CDCl ₃ ) δ 0.89 (t, J = 7.5 Hz, 3H), 1.03 (t, 3H), 1.24 (t, J = 7.1 Hz, 6H ), 2.05 (quintet, J = 7.1 Hz, 2H), 2.52 (m, 4H), 2.76-2.85 (m, 12 H), 3.35 (t, 1H), 4.13 (q, J = 7.1 Hz, 2 H), 5.28-5.44 (m, 12 H)

¹³ C NMR (75 MHz, CDCl ₃ ) δ 14.1, 14.3, 14.4, 20.5, 22.6, 25.5, 25.6, 25.7, 31.9, 44.4 , 60.1, 62.9, 127.0, 127.8, 128.05, 128.13, 128.17, 128.22, 128.5, 132.0, 173.3 (5 hidden signals)

Examples

The models and methods used in the present invention to demonstrate the effects on metabolic syndrome and type 2 diabetes are shown in Figure 2: Five blocks of experiments were performed to determine whether DHA derivatives reduce insulin resistance and / or alleviate signs of metabolic syndrome. The present invention is not limited to the presented embodiments and examples.

Example 1. Analysis of intracellular free fatty acids (unesterified fatty acids) in liver cells (block 1 in figure 2)

Background

In the first block of experiments (see figure 2), liver tissue from animals treated with PRB-1, 2, 5, and 7 were analyzed for the content of free unesterified fatty acids. Animals were recruited from the fifteenth block of experiments (pharmacodynamic effects of DHA derivatives in an animal model of metabolic syndrome). Animals received DHA (15% dietary fat) or DHA derivatives (1.5% dietary fat) for 8 weeks, and intracellular concentrations of DHA and DHA derivatives were assumed to be stable. Liver tissue was chosen because the metabolic rate in the liver is very high.

Way

Liver samples were homogenized in cold phosphate buffer solution (PBS) and immediately extracted with chloroform: methanol (2: 1) containing butylated hydroxytoluene (BHT), 0.2 mM, using cis-10-heptadecenoic acid as the internal standard. The organic phases were dried over nitrogen and redissolved in acetonitrile with 0.1% acetic acid and 10 μM BHT for analysis by reverse phase high performance liquid chromatography (RP-HPLC MS / MS). The total protein content was measured using the Bio-Rad method after homogenization.

The Agilent 1100 system was used for the reverse phase column (Supelco Ascentis C1S column, 25 cm × 4.6 mm, internal diameter 5 μm) separating DHA and its PRB derivatives for 22 minutes. The mobile phase is acetonitrile-water (87 + 13) containing 0.1% acetic acid. The column temperature was 35 ° C. For qualitative and quantitative analysis of the eluate, an ABI Qtrap-400 mass spectrometric detector with a three-stage quadrupole mass filter (ion trap) was used in the monitoring mode of multiple ions. The ion pairs were as follows: 327.3 / 327.3 (DHA), 341.3 / 341.3 (PRB-1), 355.3 / 355.3 (PRB-2 and PRB-5), 387.3 / 387.3 (PRB-7), 267.2 / 267.2 (IS FA 17: 1), respectively, under a single resolution. The exposure time was 100 ms, with the exception of FA 17: 1, where the exposure time was 200 ms. Accurate verification of PRB isomeric compounds was obtained by combining retention times and mass / charge ratios. The standard quadratic regression curve was used for quantification after determination using an internal standard.

results

The concentration of various derivatives of DHA and the concentration of DHA were presented as μg per g of total protein in the liver cells. Figure 3 shows the concentrations of various PRBs from animals that received PRB-1, 2, 5, and 7 at a concentration of 1.5% of the total fat content in a high fat diet.

The highest intracellular concentration was obtained in the case of PRB-2. Also, a high intracellular content was observed in the case of PRB-5, although not to the same extent as in the case of PRB-2. This result is unexpected.

Figure 4 shows the intracellular concentrations of DHA in liver tissues from animals that received various PRBs. DHA levels reached much higher levels in animals that received PRB-7 compared to three other DHA derivatives. Animals that received PRB-2 had the lowest concentration of DHA. PRB-7 appears to be converting back to DHA to some degree. The highest intracellular concentration was observed in the case of PRB-2. This means that the compound PRB-2 will be more accessible as a ligand for nuclear receptors, thus, a therapeutic effect will be observed, which is reflected in the control of blood glucose and blood lipids.

Example 2

Computer affinity testing (block 2 in figure 2)

Background

Nuclear receptors have been sequenced and the amino acid sequence is known for PPARs and other receptors that play a role in the genetic control of glucose and fat levels. You can use X-ray crystallography and NMR spectroscopy of PPAR receptors, and computerized testing of the affinity of fatty acids for receptors can be used to assess binding kinetics. Geometric forms of binding, often referred to as binding methods or positions, include both the position of the ligand relative to the receptor and the conformational position of the ligand and receptor. Thus, the effective ligand docking can be analyzed.

The affinity of the ligand for the receptor is determined using two different parameters: docking of the ligand (DNA derivative) to the receptor binding site and electrostatic binding between certain amino acids of the receptor and the carboxyl group or side chains of the “head” of the fatty acid (Krumrine).

As you know, the PPARα receptor is more “illegible” compared to PPARγ, that is, PPARα will take more fatty acids as ligands compared to PPARγ. However, since patients with metabolic syndrome or type 2 diabetes are usually obese or overweight and have elevated blood lipids, mainly elevated triglycerides and low levels of high density lipoprotein cholesterol, PPARα receptor activation is important. An ideal drug for treating metabolic syndrome will act as a ligand to both of these receptors, preferably with a higher affinity for the PPARγ receptor.

Way

The classification of various DHA derivatives according to their binding affinity was calculated and presented as the lowest binding affinity (LBA) and average binding affinity (ABE).

All 15 DHA derivatives (PRB-1-PRB-15) were tested using a computerized docking technique. Some of the derivatives, such as PRB-1, PRB-2, PRB-7, PRB-9, PRB-10, PRB-11, PRB-12, PRB-13, PRB-14 and PRB-15 are represented as g and s enantiomers, in which case both were tested. The PPARγ ligands rosiglitazone and pioglitazone, both in r and s form, were tested for comparison. These compounds are registered pharmaceuticals for the treatment of diabetes.

results

The results are shown in Table 1, which presents the parameters of the low binding energy of a single confirmation (LBE), the average binding energy (ABE) of a correctly oriented ICM 20 of the lowest energy confirmation (found) of the tested compounds. The affinity for RXRα was also investigated. The RXRα receptor interacts with the PPAR receptor to form a heterodimer upon binding of a fatty acid.

Figure 5 shows the binding affinity for the PPARγ receptor, which mainly plays a role in the transcription of proteins involved in the control of blood glucose. Undoubtedly, PRB-2, both in the r and s stereoisomeric form, has a strong affinity for the PPARγ receptor. PRB-5 has a lower affinity, and PRB-8 has the highest ABE. These data are highly unexpected and may result in more efficient transcription of the activated PPARγ gene, which is responsible for controlling blood glucose levels.

Figure 6 shows the affinity for the nuclear receptor PPARα, which is mainly responsible for the metabolism of fats, lipids in the blood, biology of adipose tissue and weight control. Several DHA derivatives have a high binding affinity, but the highest binding affinity is observed in the case of PRB8. It is also a highly unexpected result.

7 shows the affinity for the nuclear receptor RXRα. The physiological result of binding to the RXRα receptor has not been accurately established. It is known that RXR binds to PPAR receptors, thus forming a heterodimer, which later initiates the transcription of the described gene.

ND = no docking, c = double bonds in full-cis form. r = R enantioisomer, s = S enantioisomer. ROSI = Rosiglitazone, RIO = Pioglitazone

Some PRBs have high LBE and ABE for PPARα and PPARγ receptors, even compared to the parent DHA compound, but also compared to the PPARγ ligands of rosiglitazone and pioglitazone, both in r and s form. This interesting observation indicates that some PRBs may compete with the well-known antidiabetic drugs rosiglitazone and pioglitazone.

Ethyl derivatives in the alpha position of the same fatty acids, both in r and in s form, did not increase the degree of affinity. This has been especially confirmed for the PPARγ receptor. As already mentioned, the PPARα receptor is more “illegible” (very many fatty acids can bind). In conclusion: many tested DNA derivatives showed high affinity for the PPARα and PPARγ receptors, with a higher affinity for binding than rosiglitazone and pioglitazone.

Example 3

The study of affinity in transfected cells (block 3 in figure 2)

Background

Luciferase release correlates with gene transcription. The binding of the ligand to a nuclear receptor, such as PPARγ, induces transcription of the corresponding gene, thus, luciferase is released. This technique, therefore, provides a measurement of the affinity of the ligand for the receptor, as well as the activation of the responsible gene.

Way

Transient transfection of COS-I cells was performed in 6-well plates, as described by Graham and van der Eb (Graham). For complete transfection studies, 5 μg of the reporter construct, 2.5 μg of pSV-β-galactosidase as an internal control, 0.4 μg of pSG5-PPARγ2 were added to each well. Cells were harvested after 72 hours, luciferase activity was measured according to the protocol (Promega). Values of luciferase activity were normalized to values of β-galactosidase activity. Adipocytes were transfected with D11 differentiation using 16 μl of LipofectaminPlus reagent, 4 μl of Lipofectamine (Life Technologies Inc.), 0.2 μl of pSG5-PPARγ and 100 ng of Renilla luciferase rTK as a control of transfection efficiency. After transfection, the cells were incubated for three hours in serum containing the medium, and then incubated for 48 hours in the same medium containing the corresponding agents. Luciferase activity was measured in accordance with the manufacturer's recommendations (Dual Luciferase assay, Promega). All transfections were performed three times.

Fatty acids (BRL or DHA) and PRBs compounds (stock solutions) were dissolved in DMSO to a final concentration of 0.1 M. Then they were dissolved in 10 mM in DMSO, storage was carried out in 1.5 ml tubes (homopolymer, plastic tubes) that filled argon and stored at - 20 ° C. 10 μM compounds of PRBs or fatty acids and DMSO (control) was added to the medium 5 hours after transfection. Transfected cells were maintained in the medium for 24 hours prior to lysis with reporter lysis buffer. The binding of PRBs or fatty acids to LBD PPAR activates the binding of GAL4 to UAS, which in turn stimulates the tk promoter to trigger luciferase expression. Luciferase activity was measured using a luminometer (TD-20/20 luminometer; Turner Designs, Sunnycvale, CA) and the values were normalized to protein content.

results

The figure 8 presents the release of luciferase from transfected cells treated with various PRBs. The results indicate that PRB-1, 2, 6, 7, and 14 have a greater effect on luciferase release compared to PRB-3, 5, 9, 10, 11, 12, and 16.

Example 4

The study of the affinity for binding of PRBs and DHA in obese animals with metabolic syndrome (block 4 in figure 2)

Background

To study the affinity of PRB-2, 5, and 8 for PPARγ compared to 97% DHA and the antidiabetic compound rosiglitazone, an animal model of the metabolic syndrome (obese mice, strain C57BL / 6J) was used, the affinity study was carried out by measuring the release of luciferase from fat cells, taken from these animals. Animals (n = 8 in each group) received a high-fat diet for 8 weeks (fats comprise 60% of the total calories, the same diet was used in block 5). After that, the animals received PRBs at a dose of 1.5% of the total dietary fat for the next two weeks. A group of mice treated with rosiglitazone received 100 mg / kg of feed. Control groups continued to receive either a high fat diet or a standard diet. Figure 9 shows the research plan.

Way

After slaughtering the animals, adipose tissue (epididymal or subcutaneous) was cleared of other structures and cut into millimeter pieces. The adipose tissue was washed in 0.9% NaCl and treated in 5 ml of Krebs-Ringer solution containing Hepes, a fatty acid-free bovine serum albumin, 200 nM adenosine, 2 nM glucose and 260 IU / ml collagenase, for 1.5 hours at 37 ° C in a shaking water bath. After collagenase disruption, adipocytes were separated from debris by filtration. The cells were then washed in a Krebs-Ringer solution containing Hepes, bovine serum albumin free of fatty acids, 200 nM adenosine, 2 nM glucose, after which the cells were kept in a shaking water bath at 37 ° C for a maximum of 30 minutes before electroporation.

Isolated primary adipocytes were transfected by electroporation to measure the activity of specific proliferation-regulating elements in the peroxisome structure (PPRE). In this case, we inserted a plasmid that encodes the firefly luciferase cDNA gene under the control of PPRE from the acyl-CoA oxidase gene. In addition, cells were transfected with a plasmid containing Renilla coral luciferase cDNA under the control of a constitutively active promoter. Firefly luciferase-induced PPRE activity was normalized according to Renilla luciferase luciferase, thus, potential differences in the number of transfected cells were corrected. To measure the luciferase signal, we used the Dual-Luciferase (R) Reporter assay System (Promega, USA).

Adipose tissue was enough to isolate adipocytes for duplicates. The animals of each group were slaughtered at 2-day intervals and 4 independent transfections were obtained for each diet group.

results

Over the first 8 weeks, mice fed a high-fat diet (HF diet) (33.7% fat, w / w) showed a gradual increase in body weight compared to control mice that received a regular diet (4.5 % w / w). Over the next two weeks, animals fed a high fat diet and animals fed a high fat diet in combination with rosiglitazone continued to gain weight at approximately the same rate as before. With a high fat diet supplemented with PRB-8 and PRB-5, the rate of weight gain was reduced. However, in the case of obtaining PRB-2 and DHA (5% w / w), the weight gain completely stopped, and the body weight even decreased (Fig. 10). Food intake was monitored from time to time (4x). In this aspect, there were no differences between the groups receiving the high fat diet and the groups receiving the PRB compounds.

In the case of obtaining a diet high in fat in combination with rosiglitazone, the endogenous activity of the PPARγ receptor was approximately 2 times higher than in all other groups receiving a specific diet (Fig. 11). In addition, these fat cells became more sensitive to additional in vitro stimulation of 5 uM rosiglitazone (stimulation by 5, 12 times) compared to the diet with a high fat content (stimulation by 1.5 times). This rosiglitazone sensitizing effect was also observed in the groups receiving PRB-2 and PRB-5 (stimulation 2.6 times).

The data obtained in this study clearly showed activity against nuclear receptors of PPAR, in particular with regard to effects on body weight, the most noticeable results were obtained with groups receiving compound PRB-2. Even animals treated with PRB-5 and PRB-8 did not gain weight as fast as animals in the group that received only a high-fat diet. Interestingly, in animals receiving rosiglitazone, body weight increased to the same extent as in animals receiving only a high-fat diet. This clearly demonstrates the negative effects of using only PPARγ ligands, such as glitazones, since there is a risk of increasing body weight, even if insulin resistance is reduced. However, when it comes to activating PPARγ, measured in this experiment as luciferase activity, the effect of rosiglitazone is more pronounced than the effect of any of the compounds of PRBs. Compounds PRB-2 and PRB-5 were more effective than PRB-8 and DHA (Fig. 12).

Example 5

Pharmacodynamic effects of DHA derivatives in an animal model of metabolic syndrome (block 5 in figure 2)

Background

An animal model of metabolic syndrome (mice prone to obesity, strain C57BL / 6J) was used to demonstrate effects on typical laboratory and pathological anatomical features characteristic of metabolic syndrome. When receiving a diet high in fat (60% fat), animals developed obesity, plasma insulin levels increased significantly, glucose tolerance was impaired, serum levels of triglycerides and non-esterified fatty acids increased, and liver obesity was observed.

Example 5a

Effects of DHA derivatives in mice prone to obesity during a 4-month experimental diet.

Way

All experiments were performed on male mice of strain C57BL / 6, sub-strain C57BL / N (supplier: Charles River, Germany, n = 160, experiments AC, see below) or sub-strain C57BL / 6J (supplier: the Jackson laboratory, Bar Harbor, ME, USA, n = 32, experiment D). The total number of animals used was higher due to culling (n = 170 and 36, respectively). In the latter case, animals were bred for several generations (<20) at the Institute of Physiology. At the beginning of the experiment, the animals were 14 weeks old and their weight was 23.6-27.1 g. A week before the start of the study, the animals were sorted into 8 subgroups depending on their body weight. About 5-10% of the animals with the smallest and largest weight, respectively, were rejected. Animals excluded from the study at this stage were euthanized by cervical dislocation. A complete health check of the mice was performed by the Charles River supplier company and at the start of the studies, serological tests were performed by ANLAB laboratory (Prague, Czech Republic). In addition, regular health checks were performed in special rooms for animals at 3-month intervals when using indicator animals, serological studies (ANLAB) were conducted. Studies have shown that animals were free of infections.

Diets

Animals received three types of experimental diets:

(i) Mixed diet (ssniffRM-H from SSNIFF Spezialdieten Gmbh, Soest, Germany; see also http://ssniff.de), in which proteins, fats and carbohydrates accounted for 33, 9 and 58% of the energy, respectively.

(ii) A high fat diet (cHF diet), prepared in the laboratory, in which proteins, fats and carbohydrates accounted for 15, 59 and 26% energy, respectively, a well-characterized fatty acid composition was added to this diet (most lipids were isolated from corn oil; see Ruzickova 2004).

(iii) Laboratory-based high-fat diets in which 0.15, 0.5, and 1.5% fat (a corn oil component) have been replaced with various PRB compounds (PRB1, PRB2, PRB5, PRB7, and PRB8) or DHA. All of these compounds were in the form of ethyl esters obtained from Pronova Biocare in sealed containers. The chemical composition of the PRB compounds was unknown to laboratory workers conducting experiments (Institute of Physiology. Prague Academy of Sciences, Czech Republic).

After receiving the connection, PRB was stored in the refrigerator in the original containers. The containers were opened immediately before the preparation of the experimental diets. The diets were stored in plastic bags filled with nitrogen, and stored at -70 ° C in small portions, which were enough to feed the animals for a week. Fresh rates were given at 2-day intervals or daily.

Research plan

The study included 4 experiments. In each experiment, various PRB (or DHA) compounds were mixed with a high fat diet in three different concentrations (0.15, 0.5, and 1.5% of the total fat content). In each experiment, a subset of mice that served as controls was included. Mice were placed in cells of 4 each and received a mixed diet up to 3 months of age, then the animals (n = 8-13) were randomly divided into groups that received different experimental diets. After two months of receiving a specific diet (at 5 months of age), the animals were left without food during the night and in the morning and a glucose tolerance test (intraperitoneal injection) was performed. Animals were slaughtered at 7 months of age after 4 months of an experimental diet.

Study Parameters

The parameters in the study were as follows: weight gain (grams), area under the curve (AUC) in glucose tolerance tests (mmol × 180 min), plasma insulin level (ng / ml), serum triglycerides (TAGs mmol / 1 ) and unesterified fatty acids (NEFA mmol / 1).

On Fig shows a typical curve of the clearance of glucose from the blood before and after giving animals a compound that reduces insulin resistance. A decrease in the area under the curve indicates that the blood glucose level decreases faster due to reduced insulin resistance.

results

The results are presented in tables 2, 3 and 4. (* = significant differences compared with diets high in fat (P <0.05).)

Table 2 shows the effects in animals that received test compounds of PRB at a concentration of 1.5% compared with animals that received a diet high in fat or 97% DHA. The rate of weight gain was significantly reduced in animals treated with PRB-2 compared to animals fed a high fat diet (cHF). In this group, food intake was slightly reduced. The most pronounced decrease in the area under the curve in the glucose tolerance test was observed in the same group and even in animals treated with PRB-1. Plasma insulin levels were significantly lower in the PRB-2 group compared to cHF controls, even if some animals also showed some effect on this parameter in animals treated with PRB-1 and PRB-5. In the group treated with PRB-2, there was the greatest decrease in triglycerides (TAGs) and unesterified fatty acids (NEFA).

Table 3 shows the effects in animals that received the lowest concentration, 0.5%, of the test compounds compared to animals that received a standard diet, a high fat diet (cHF), or 97% DHA. Weight gain was slightly lower in animals treated with PRB-2 and PRB-5. The AUC in the glucose tolerance test, as well as the plasma insulin level, were, however, significantly lower only in the group treated with PRB-2.

Table 4 presents the results of the diet with the lowest concentration of PRB, 0.15%. In this case, the differences were very small. The weight gain was slightly lower in the groups treated with PRB-1 and PRB-2, while the AUC was significantly lower only in the group treated with PRB-2. Plasma insulin levels were lower in the groups treated with PRB-1, 2, and 7.

table 2 The effect of derivatives of PRB after a 4-month diet containing 1.5% PRB Parameter STD cf PRB-1 PRB-2 PRB-5 PRB-7 Dha Body weight (grams) 32.4 ± 0.7 49.6 ± 0.6 44.0 ± 1.5 * 30.1 ± 1.1 * 46.3 ± 1.6 45.9 ± 1.1 * 47.1 ± 0.7 * Weight gain (grams) 7.8 ± 0.4 25.2 ± 0.5 20.2 ± 1.3 * 6.4 ± 0.8 * 22.4 + 1.4 21.7 ± 0.9 * 23.0 ± 0.8 * Food intake (grams / mouse / day) 3.64 ± 0.04 2.70 ± 0.02 2.64 ± 0.03 2.38 ± 0.05 * 2.62 ± 0.02 2.68 ± 0.03 2.63 ± 0.02 AUC glucose (mm × 180min) 1124 ± 57 1625 ± 151 913 ± 68 * 982 ± 89 * 1264 ± 192 1122 ± 73 2132 ± 288 * Fasting glucose (mg / dl) 77 ± 3 145 ± 7 130 ± 14 95 ± 6 * 136 ± 12 120 ± 9 138 ± 7 Insulin (ng / ml) 1.03 ± 0.09 5.35 ± 0.36 2.73 ± 0.33 0.60 ± 0.18 * 2.47 ± 0.19 * 4.42 ± 0.87 6.55 ± 0.31 Triglycerides (mmol / L) 1.41 ± 0.09 1.45 ± 0.07 1.58 ± 0.08 0.71 + 0.01 * 1.19 ±, 0.07 1.15 ± 0.08 1.91 ± 0.26 * Unesterified fatty acids (nmol / L) 0.57 ± 0.05 0.61 ± 0.04 0.63 ± 0.03 * 0.54 ± 0.03 * 0.72 ± 0.05 0.82 ± 0.06 0.98 ± 0.07

Table 3 The effect of derivatives of PRB after a 4-month diet containing 0.5% PRB Parameter STD cHF PRB-1 PRB-2 PRB-5 PRB-7 Dha Body weight (grams) 32.4 ± 0.7 49.6 ± 0.6 47.4 ± 0.6 45.8 ± 1.7 45.7 ± 1.5 48.8 ± 0.9 46.9 ± 0.7 * Weight gain (grams) 7.8 ± 0.4 25.2 ± 0.5 23.8 ± 0.5 21.9 ± 0.6 22.0 ± 1.4 24.8 ± 0.8 22.9 ± 0.7 * Food intake (grams / mouse / day) 3.64 ± 0.04 2.70 ± 0.02 2.67 ± 0.04 2.69 ± 0.04 2.63 ± 0.02 2.69 ± 0.03 2.70 ± 0.03 AUC glucose (mm x 180 min) 1124 + 57 1625 ± 151 1596 + 205 1224 ± 72 * 1581 ± 231 1674 ± 203 1816 ± 182 Fasting glucose (mg / dl) 77 ± 3 145 ± 7 131 ± 7 136 ± 7 130 ± 7 152 ± 6 136 ± 8 Insulin (ng / ml) 1.03 ± 0.08 5.35 ± 0.36 3.93 ± 0.59 2.75 ± 0.21 * 5.12 ± 0.93 4.10 ± 0.57 * 5.82 ± 0.47 Triglycerides (mmol / L) 1.41 ± 0.09 1.45 ± 0.07 2.03 ± 0.22 1.29 ± 0.08 1.4b ± 0.17 1.42 ± 0.08 1.78 ± 0.08 * Unesterified fatty acids (nmol / L) 0.57 ± 0.05 0.61 ± 0.04 0.73 ± 0.04 * 0.75 ± 0.04 0.77 ± 0.03 * 0.87 ± 0.04 0.89 ± 0.03

Table 4 The effect of derivatives of PRB after a 4-month diet containing 0.15% PRB Parameter STD cHF PRB-1 PRB-2 PRB-5 PRB-7 Dha Body weight (grams) 32.4 ± 0.7 49.6 ± 0.6 47.2 ± 1.3 46.7 ± 1.1 48.0 ± 0.8 47.4 ± 0.8 * 48.3 ± 0.6 Weight gain (grams) 7.8 ± 0.4 25.2 ± 0.5 22.9 ± 1.1 22.8 ± 0.9 24.2 ± 0.5 23.2 ± 0.7 * 24.3 ± 0.8 Food intake (grams / mouse / day) 3.64 ± 0.04 2.70 ± 0.02 2.63 ± 0.04 2.57 ± 0.03 * 2.66 ± 0.02 2.59 ± 0.02 2.79 ± 0.03 AUC of glucose (mm × 180 min) 1124 ± 57 1625 ± 151 1291 ± 172 1071 ± 148 * 1443 ± 70 1425 ± 97 1477 ± 214 Fasting glucose (mg / dl) 77 + 3 145 ± 7 126 ± 15 132 ± 5 151 + 5 141 ± 9 141 ± 10 Insulin (ng / ml) 1.03 ± 0.08 5.35 ± 0.36 3,50 ± 0,29 4.00 ± 0.64 6.21 ± 0.45 3.76 ± 0.72 * 4.31 ± 0.39 * Triglycerides (mmol / L) 1.41 ± 0.09 1.45 ± 0.07 1.75 ± 0.08 1.42 ± 0.07 1, b4 ± 0.28 1.41 ± 0.11 1,50 ± 0,13 Unesterified fatty acids (nmol / L) 0.57 ± 0.05 0.61 ± 0.04 0.62 ± 0.04 * 0.78 ± 0.04 * 0.71 ± 0.09 0.85 ± 0.06 0.96 ± 0.07

In conclusion: a study of the use of PRB-1, 2, 5, and 7 for 4 months in obese animals with insulin resistance and metabolic syndrome showed an obvious and unexpected effect of tested PBRs and, in particular, the DHA derivative of PRB-2 on resistance to insulin and the symptoms of metabolic syndrome, weight loss, a decrease in AUC in the intraperitoneal glucose tolerance test, and a decrease in triglycerides and unesterified fatty acids were observed. Effects were observed in the group receiving a dose of PRB of 1.5%, and the group receiving a dose of 0.5%. Some effects were noted even in the group receiving the lowest concentration of 0.15%.

The test of compound PRB-8 began later, therefore, in three groups receiving a diet with a concentration of this compound 1.5%, 0.5% and 0.15%, only 2-month studies were obtained. In the group receiving 1.5%, body weight (BW) was 28.0 ± 0.7 g compared with the control 29.6 ± 0.9, AUC was 1031 ± 104 compared with the control 1074 ± 91. These differences are small, but the trend is interesting. For these parameters, there was no difference between experimental diets in which animals received the lowest doses of 0.5% and 0.15%, and control groups. Data obtained with PRB-8 (application within 2 months) showed that there is a tendency to weight loss and AUC.

Example 5b

Effect of DHA Derivatives on Metabolic Syndrome and Insulin Resistance

Way

In another experiment, PRB-2, PRB-5, and PRB-7 were tested on animals of the same breed. In this experiment, the animals initially received a high-fat diet (as in the previous experiment 5a) for 8 weeks, the animals developed insulin resistance and metabolic syndrome, and then they received compounds of PRBs. The initial dose of PRBs was 15% fat, but animals did not tolerate this dose. After a 2-week period, animals received 1.5% PRB-2, 5% and 1.5% PRB-5, and 1.5% and 0.5% PRB-7.

results

Weight loss was very pronounced in animals treated with PRB-2. Even in animals treated with PRB-5, some weight loss was observed, but at a higher dose - 5%. Triglycerides were reduced when all tested derivatives were used compared to control animals fed a high fat diet (cHF diet). The decrease in the level of unesterified fatty acids was more pronounced with the use of PRB-2 and PRB-5, however, in different doses (see Fig. 14).

Blood cholesterol levels were reduced in animals treated with PRB-2 and PRB-5. The blood glucose level was not changed due to the fact that these animals are in a prediabetic state with a normal glucose level due to the high level of secretion of insulin production. However, the most important fact is that plasma insulin levels were significantly reduced in the group of animals treated with PRB-2 at a much lower concentration compared to the second very effective DHA derivative PRB-5. Even with PRB-7, effects on insulin concentration were observed (see FIG. 15).

When using PRB-2, a statistically significant decrease in blood glucose AUC was observed at all time points compared with the initial values. This means that blood glucose levels decreased after applying PRB-2 at a concentration of 1.5%. When using PRB-5 and PRB-7, some effect was observed, but to a lesser extent than in the case of PRB-2 (see Fig. 16).

These results are very unexpected and suggest a positive effect of these compounds in metabolic syndrome and type 2 diabetes. Almost all of these patients are obese or overweight, and the results showed that the drug is promising in terms of weight loss. The most commonly used treatments for type 2 diabetes today are thiazolidinones, which are strong PPARγ ligands, thus reducing insulin resistance, which often leads to increased body weight, which is very undesirable for these patients (Yki-Jarvinen 2004) .

Lowering serum triglycerides is another very important effect that was shown in these experiments. In patients with metabolic syndrome and type 2 diabetes, triglycerides are often elevated. The effects of DHA derivatives in lowering triglycerides are a very positive result and again the PRB-2 compound showed the greatest effect at the lowest doses. Very positive effects on plasma insulin levels and glucose tolerance test are very promising and very unexpected. Taken together, all the results obtained with DHA derivatives, especially PRB-2, are very encouraging to form a good base for the development of an antidiabetic drug.

Example 5c

The use of derivatives of DHA for fatty degeneration of the liver

Way

In experiments with DHA derivatives, tissue samples were taken and histological examination was performed. After waxing, samples of liver tissue, adipose tissue, skeletal muscle, pancreas, and kidneys were stained with eosin-hematoxylin.

results

No pathological changes in tissues except liver tissue were detected. In control animals that received a high-fat diet, fatty liver was observed (liver steatosis). Fat droplets in the liver were easily distinguishable from normal liver cells. In animals treated with compounds PRB-1, 5 and 7, a low degree of fatty degeneration of the liver was noted. However, in animals treated with PRB-2 at a concentration of 1.5%, no signs of fatty degeneration of the liver were observed. This observation is extremely important and of great importance for the treatment of patients with insulin resistance, obesity and type 2 diabetes. Fatty degeneration of the liver is a very common occurrence in these patients, which is associated with an excess of fatty acids and triglycerides, biological markers in the development of insulin resistance and metabolic syndrome. Derivatives of DHA reduce fatty degeneration of the liver, the most pronounced effect was shown by the compound PRB-2.

DISCUSSION AND CONCLUSION

This application contemplates a new group of compounds that activate nuclear receptors, especially PPARγ and PPARα, thus exerting a number of therapeutic effects in the treatment of insulin resistance, metabolic syndrome, type 2 diabetes, cardiovascular diseases and other diseases associated with atherosclerosis.

Members of this group are derivatives of DHA with various side chains in the alpha position of the molecules. A large number of alpha-substituted DHA derivatives have been tested and compared with controls, as well as with pure DHA and EPA. Some tested compounds have shown interesting biological effects relevant to the development of an antidiabetic drug based on them.

An interesting and yet incomprehensible fact is that alpha-ethyl-DHA ethyl ester (PRB-2) was significantly more effective in a number of tests used to demonstrate the effects of insulin resistance and, as a result, with diseases caused by this pathophysiological condition , these diseases are metabolic syndrome, type 2 diabetes, cardiovascular diseases and other diseases associated with atherosclerosis. The liver tissue of animals that received various derivatives of DHA (block 1) was saturated with alpha-ethyl-DHA ethyl ester, indicating that this compound was not used to synthesize triglycerides, eicosanoids, or other metabolic intermediates. Indirectly, this means that alpha-ethyl-DHA will be available for binding to nuclear receptors such as PPARs.

Testing the affinity for PPARγ and PPARα using docking technology showed that a large number of DHA derivatives have affinity for both receptors and last but not least for PPARγ, which is probably the most important nuclear receptor involved in activating the genes responsible for glucose metabolism in blood. Especially alpha-ethyl-DHA (PRB-2) and alpha-diethyl-DHA (PRB-8) had a strong affinity for these nuclear receptors. Compared to alpha-diethyl-DHA, alpha-ethyl-DHA has two stereoisomers, r and s forms. Using docking technology, it was shown that both stereoisomers have the same affinity for PPARγ and PPARα, which means that neither the r nor s form have advantages over the racemic form. Moreover, the racemic form may have advantages over each of the isomers.

When affinity was investigated in transfected cells carrying a nuclear receptor and a DNA response element, some PRBs were shown to have strong affinity, measured as luciferase release. The best results were observed in the case of alpha-ethyl-DHA (PRB-2) and PRB-6, 7 and 14.

Five DHA derivatives were tested in mice of strain C57BL / 6, which developed insulin resistance and metabolic syndrome when receiving a high-fat diet. The alpha-ethyl-DHA compound (PRB-2) was tested in three separate experiments, while the PRB-1, 5 and 7 were tested in two experiments and the alpha-diethyl-DHA compound (PRB-8) was tested in one experiment. All derivatives showed significant biological effects. However, in the case of alpha-ethyl-DHA (PRB-2), the most promising effects were observed: a decrease in body weight, AUC in the intraperitoneal glucose tolerance test, plasma insulin levels, as well as a decrease in the concentration of triglycerides and unesterified fatty acids in blood serum. These results were obtained at doses of 1.5% and 0.5%. At the lowest dose of 0.15%, the effects were not pronounced. The alpha-ethyl-DHA (PRB-2) compound at a dose of 1.5% was also effective in preventing fatty liver disease, which often develops in humans and animals with insulin resistance and metabolic syndrome.

The compound alpha-ethyl-DHA (PRB-2) acts 10-30 times stronger than pure DHA. The results of comparing the derivatives of DHA with the original DHA molecule in terms of potency were highly unexpected.

Since alpha-ethyl-DHA (PRB-2) seems to work by binding to the nuclear receptors PPARγ and PPARα, the compound not only has interesting therapeutic effects on the metabolism of carbohydrates and fats, not least in patients with insulin resistance, metabolic syndrome and type 2 diabetes, but also reduces weight and has a general anti-inflammatory effect. The alpha-ethyl-DHA (PRB-2) compound, directly or through exposure to risk factors, can prevent the development of cardiovascular diseases such as myocardial infarction and cerebrovascular accident, and this compound can also reduce cardiovascular mortality.

Pharmaceutical agents acting as PPARγ ligands are already on the pharmaceutical market, but even if these compounds have a positive effect on carbohydrate metabolism, they have side effects such as increased triglycerides, increased body weight, and swelling. The alpha substituted DHA derivatives provided herein have a combined effect on the PPARγ and PPARα receptors, which may be advantageous for patients with insulin resistance, metabolic syndrome and type 2 diabetes. In addition, this combined effect affects blood lipids, inflammation, atherosclerosis and, consequently, cardiovascular disease.

The present invention is not limited to the shown embodiments and examples.

Information sources

Simonopoulos AR. Essential fatty in health and chronic disease. Am J ClinNutr 1999; 70 (Suppl): 560S-569S.

Geleijnse JM, Giltay EJ, Grobbee DE, et al. Blood pressure response to fish oil supplementation: metaregression analysis of randomized trials. J Hypertension 2002; 20: 1493-1499.

Storlien LH, Hulbert AJ, and Else PL. Polyunsaturated fatty acids, membrane function and metabolic diseases such as diabetes and obesity. Curr Opin Clin Nutr Metab Care 1998; 1 - 559-563.

Jump DB. The biochemistry of n-3 polyunsaturated fatty acids, J Biol Chem 2002; 277-8755-8758.

Pawar A and Jump D. Unsaturated fatty acid regulation ofperoxisomes proliferator-activated receptor alfa activity in rat primary hepatocytes. J Biol Chem 2003; 278: 35931-35939.

Meigs JB, Wilson PWF, Nathan DM, et al. Prevalence and characteristics of the metabolic syndrome in the San Antonio Heart and Framingham offspring studies. Diabetes 2003; 52: 2160-2167.

Storlien LH, KraegenWE, Chisholm DJ, et al. Fish oil prevents insulin resistance induced by high fat feeding in rats. Science 1987; 237: 885-888.

Field CJ, Ryan EA, Thomson ABR, et al. Diet fat composition alters membrane phospholipid composition, insulin binding and glucose metabolism in adipocytes from control and diabetic animals. J Biol Chemistry 1990; 265: 11143-11150.

Yki-Jarvinen, H. Thiazolidinediones. NEJM 2004; 351: 1106-1118. Adams M, Montague CT5 Prins JB, et al. Activators of peroxisomes proliferator-activated receptor gamma have depot-specific effects on human preadipocyte differentiation. J Clin Invest 1997, 100: 3149-3153.

Ruzickovaj, Rossmeisl M, Prazak T, et al. Omega-3 PUFA of marine origin limit diet-induced obesity in mice by reducing cellularity of adipose tissue, Lipids 2004; 39: 1177-1185.

Vaagenes H, Madsen L, Dyroy E, et al. The hypolipidaemic effect of EPA is potentiated by 2- and 3-methylation. Biochim Pharmacol 1999; 58: 1133-1143.

Larsen L, Granslund L, Holmeide AK, et al. Sulfur-substituted and [alpha] -methylated fatty acids as peroxisome proliferator-activated receptor activators. Lipids 2005, 40: 49-57.

Larsen L, Horvik K, Sorensen HIN, et al. Polyunsaturated thia- and oxa-fatty acids: incorporation into cell-lipids and their effects on arachidonic acid- and eikosanoids synthesis. Biochim et Biophys Acta 1997, 1348: 346-354.

Larsen, et.al. Biochemical Pharmacology 1998; 55, 405.

Chih-Hao L, Olson P, and Evans RM. Lipid metabolism, metabolic diseases, and peroxisome proliferator-activated receptors. Endocrinology 2003; 144: 2201-2207.

Willumsen N, Waagenes H, Holmsen H, et al. On the effect of 2-deuterium- and 2-methyl-eicosapentaenoic acid derivatives on triglycerides, peroxisomal beta-oxidation and platelet aggregation in rats. Biochim Biophys Acta 1998; 1369: 193-203.

Mitsunobu O, Synthesis 1981; 1.

Ager DJ, Prakash I, and Schaad DR. 1,2-amino alcohols and their heterocyclic derivatives as chiral auxiliaries in asymmetric synthesis Chem Rev 1996; 96: 835-876.

Claims (61)

1. The compound of the formula

(4Z, 7Z, 10Z, 13Z, 16Z, 19Z) -ethyl 2-ethyl docosa-4,7,10,13,16,19-hexanoate. 2. The compound according to claim 1, characterized in that the compound is present as the S enantiomer. 3. The compound according to claim 1, characterized in that the compound is present as the R enantiomer. 4. The compound according to claim 1, characterized in that the compound is present as a mixture of R and S enantiomers. 5. The compound according to claim 4, characterized in that the mixture is racemic. 6. A composition for treating diabetes, for reducing insulin, blood glucose, serum triglyceride, for treating dyslipidemia, for lowering blood cholesterol, for reducing body weight, and for treating peripheral insulin resistance, comprising an effective amount of a compound according to claim 1 and at least one pharmaceutically acceptable excipient. 7. The composition according to claim 6, formulated for oral administration. 8. The composition according to claim 6 in the form of a capsule or sachet powder. 9. The composition according to claim 6, formulated for intravenous, subcutaneous or intramuscular administration. 10. The composition according to claim 6, formulated in such a way that it provides a daily dose of 10 mg to 10 g. 11. The composition of claim 10, formulated so that it provides a daily dose of from 100 mg to 1 g of the compound. 12. A food lipid composition comprising a pharmaceutically effective amount of a compound according to claim 1. 13. The food lipid composition according to item 12, wherein the compound according to claim 1 is at least 60% of the total weight of the composition. 14. The food lipid composition of claim 13, wherein the compound of claim 1 is at least 90% of the total weight of the composition. 15. The food lipid composition according to item 12, wherein the composition further comprises a pharmaceutically acceptable antioxidant. 16. The food lipid composition according to clause 15, wherein the pharmaceutically acceptable antioxidant is tocopherol. 17. A method of treating and / or preventing diabetes, comprising administering to a human or animal a pharmaceutically effective amount of a compound according to claim 1. 18. The method according to 17, characterized in that the diabetes is type 2 diabetes. 19. A method of reducing insulin, blood glucose and / or serum triglyceride, comprising administering to a human or animal a pharmaceutically effective amount of a compound according to claim 1. 20. A method of treating and / or preventing dyslipidemia, comprising administering to a human or animal a pharmaceutically effective amount of a compound according to claim 1. 21. The method according to claim 20, characterized in that dyslipidemia is a hyperlipidemic state. 22. The method according to item 21, wherein the hyperlipidemic state is hypertriglyceridemia. 23. A method of lowering blood cholesterol, comprising administering to a human or animal a pharmaceutically effective amount of a compound according to claim 1. 24. A method of reducing body weight and / or preventing weight gain, comprising administering to a human or animal a pharmaceutically effective amount of a compound according to claim 1. 25. A method of treating or preventing peripheral insulin resistance, comprising administering to a human or animal a pharmaceutically effective amount of a compound according to claim 1. 26. The compound of formula (I)

characterized in that X is a carboxylic acid, carboxylate, carboxylic anhydride, diglyceride, triglyceride, phospholipid or carboxamide,
or any pharmaceutically acceptable salt thereof. 27. The compound of claim 26 of formula (II)

28. The compound of claim 26 in salt form, wherein X is COO ^- Z ⁺ , where Z ⁺ is Li ⁺ , Na ⁺ , K ⁺ , NH ₄ ⁺ or substituted NH ₄ ⁺ . 29. The compound of claim 26 in the form of a salt of the formula

where Z ²⁺ is Mg ²⁺ or Ca ²⁺ . 30. The compound according to claim 26, wherein the carboxylate group is selected from ethyl carboxylate, methyl carboxylate, n-propyl carboxylate, isopropyl carboxylate, n-butyl carboxylate, sec-butyl carboxylate and n-hexyl carboxylate. 31. The compound according to p. 26, wherein the carboxamide group is selected from primary carboxamide, N-methylcarboxamide, N, N-dimethylcarboxamide, N-ethylcarboxamide and N, N-diethylcarboxamide. 32. The compound according to p, characterized in that the carboxylate group is methyl or ethyl carboxylate. 33. The compound according to p. 26, characterized in that the compound is present as the S enantiomer. 34. The compound according to p. 26, wherein the compound is present as the R enantiomer. 35. The compound according to p. 26, characterized in that the compound is present as a mixture of R and S enantiomers. 36. The compound according to clause 35, wherein the mixture is racemic. 37. A composition for treating diabetes, for reducing insulin, blood glucose, serum triglyceride, for treating dyslipidemia, for lowering blood cholesterol, for reducing body weight, and for treating peripheral insulin resistance, comprising an effective amount of a compound 26, and at least one pharmaceutically acceptable excipient. 38. The composition according to clause 37, further comprising a compound of the formula

39. A method of treating and / or preventing a disease or condition selected from peripheral insulin resistance / diabetic condition; and obesity or overweight; comprising administering to a human or animal a pharmaceutically effective amount of a compound according to claim 26. 40. The method according to § 39, wherein the diabetic condition is type 2 diabetes. 41. A method for the treatment and / or prevention of dyslipidemia, comprising administering to a human or animal a pharmaceutically effective amount of a compound according to claim 26. 42. The method according to paragraph 41, wherein the dyslipidemia is a hyperlipidemic state. 43. The method of claim 42, wherein the dyslipidemia comprises elevated levels of triglyceride and / or non-HDL cholesterol (LDL cholesterol and VLDL cholesterol). 44. A method for reducing insulin, blood glucose and / or serum triglyceride by administering to a human or animal a pharmaceutically effective amount of a compound according to claim 26. 45. A method of reducing body weight and / or preventing weight gain by administering to a human or animal a pharmaceutically effective amount of a compound according to claim 26. 46. A method for activating and / or binding isoforms of at least one human peroxisome proliferation activating receptor (PPAR) by administering to a human or animal a pharmaceutically effective amount of a compound according to claim 26. 47. The pharmaceutical composition according to clause 37, formulated for oral administration. 48. The pharmaceutical composition according to clause 37 in the form of a capsule or sachet powder. 49. The pharmaceutical composition according to clause 37, formulated for intravenous, subcutaneous or intramuscular administration. 50. The pharmaceutical composition according to clause 37, designed in such a way that it provides a daily dose of from 10 mg to 10 g 51. The pharmaceutical composition according to clause 37, formulated in such a way that provides a daily dose of from 100 mg to 1 g 52. A food lipid composition comprising a pharmaceutically effective amount of a compound according to claim 26 and at least one pharmaceutically acceptable excipient. 53. The food lipid composition according to paragraph 52, wherein the compound of formula (I) is at least 60% of the total weight of the composition. 54. The food lipid composition according to item 53, wherein the compound of formula (I) is at least 90% of the total weight of the composition. 55. The food lipid composition according to paragraph 52, further comprising a pharmaceutically acceptable antioxidant. 56. The food lipid composition of claim 55, wherein the pharmaceutically acceptable antioxidant is tocopherol. 57. A method of producing a compound of ethyl (all-Z) -2-ethyl docosa-4,7,10,13,16,19-hexanoate

including:
a) reacting the solution containing the DHA ester with a strong non-nucleophilic base to produce ester enolate, and
b) reacting the enolate obtained in step a) with an electrophilic ethyl reagent representing ethyl iodide;
c) extracting the product from the solution obtained in step b) with a solvent. 58. The method according to clause 57, wherein the DHA ester is obtained from a source of plant, microbial, animal origin, or combinations thereof. 59. The method according to clause 57, wherein the DHA ester is obtained from sea fat. 60. The method according to § 59, wherein the sea oil is fish oil. 61. The method according to clause 57, wherein the strong non-nucleophilic base is selected from lithium diisopropylamide, potassium hexamethyldisilazane and sodium hexamethyldisilazane.

Images