Fatty acid derivatives of bile acids and bile acid derivatives and pharmaceutical compositions thereof

Description

FATTY ACID DERIVATIVES OF BRE ACIDS! AND OTHER BILE ACID DERIVATIVES The present invention relates to bile acids, or bile acid conjugates with fatty acids (hereinafter referred to as "BAFAC"), the use of these compounds for dissolving cholesterol-based gallstones in the bile, preventing their appearance and recurrence, uses for reducing or preventing atherosclerosis, and methods for treating said diseases. It should be noted that the terms bile acid and bile are similar in meaning, and therefore they are used interchangeably. In most industrialized countries, gallstones occur in about 15% of people. Most gallstones are cholesterol-based, meaning their main component is cholesterol. Thus, cholesterol-based gallstones are a serious health problem. Bile is often supersaturated with cholesterol, and this cholesterol is prone to crystallizing. Preventing cholesterol from crystallizing in bile can prevent the formation of cholesterol-based gallstones or their recurrence after gallstone crushing, dissolution, or gallstone removal procedures. The residence time of newly secreted bile in the gallbladder is short - less than 12-24 hours. During this time, preventing cholesterol from crystallizing can prevent the formation of gallstones. It has been shown that cholesterol-based gallstones can be medically dissolved and their recurrence can be prevented by the use of certain bile acids, such as chenodeoxycholic acid or ursodeoxycholic acid. However, bile acid therapy is of low efficacy and very time-consuming, and is therefore often abandoned. Therefore, more effective treatments are needed. Recent research has demonstrated the important role of phospholipids in the dissolution of cholesterol in bile (T. Gílat et al., Biochimica and Biophysica Acta 1286, (1996), 96-115; Y. Ringel et al., Biochimica and Biophysica Acta 1390, (1998), 293-300; and 3. Hepatology, 28, (1998), 1008-1014). Phospholipids are the sole or main components of the fat particles containing dissolved cholesterol in bile. It has been shown that the gradual addition of phospholipids to bile gradually prolongs the time for the cholesterol content of bile to crystallize (Z. Halpern et al., Gut 34 (1993) 110-115). It has been shown that certain phospholipid molecules have large differences in their ability to inhibit cholesterol crystallization in human or model bile. Phospholipids differ from each other mainly in the stereospecific sn-1 and / or sn-2 positions of the fatty acids present in them, as well as in their head groups. It has been shown that the formation of cholesterol crystal precipitates can be significantly delayed and the growth rate of cholesterol crystals and the total mass of cholesterol crystals can be greatly reduced by changing the composition of the phospholipid molecule without changing the absolute or relative amount of phospholipids. Cholesterol crystallization is greatly delayed if the sn-2 fatty acid is saturated, if the head group is serine instead of choline, etc. (Y. Ringd et al., supra). It has also been shown that various components of phospholipids, such as saturated fatty acids such as palmitic acid or stearic acid; or phosphatidylglycerol, alone (without the entire phospholipid molecule) have a strong inhibitory effect on cholesterol crystallization. Thus, increasing the amount of phospholipids in human bile in general, or the amount of certain phospholipids or their components, such as fatty acids, significantly delays the crystallization of cholesterol in bile, thereby achieving the desired result. The problem is how to enrich the amount of phospholipids or their components in the bile in a living organism. When bile salts are administered to humans, they are very efficiently absorbed and excreted in the bile. This also applies to synthetically produced bile salt derivatives. The body has well-developed and very efficient transport mechanisms for this purpose. Thus, if ursodeoxycholic acid (which is normally present in very small amounts in human bile) is administered regularly, it is bound and excreted in the bile, eventually constituting 30-50% of the bile acid content of bile. However, as indicated above, bile therapy for the dissolution of cholesterol-based gallstones is not satisfactory. The liver binds and takes up phospholipids and their components. However, the secretion of phospholipids into the bile is tightly regulated by the liver, and only a limited amount and type of phospholipids are transferred into the bile, in conjunction with the secretion of bile salts and cholesterol. At present, there is no effective method that can modify the phospholipid composition of human bile to any appreciable extent, either quantitatively or qualitatively. When dietary phospholipids reach the liver, the liver modifies them, secretes them into the blood, or stores them in the liver. Only small amounts and predetermined types of substances are secreted into the bile, and there is little possibility of modifying them. Therefore, it was important to find a satisfactory method for delivering phospholipids, or one of their components, into the bile, which would improve the dissolution of cholesterol in the bile and prevent the formation of cholesterol-based gallstones or promote the dissolution of existing gallstones. From Israeli patent application No. Á 95688 and the corresponding United States patent applications, bile acid derivatives of the general formula (I) are known: W - X - G in which formula G is a bile acid radical, W is an active group of a medicinal compound, X is either a direct bond or a linking group between the bile acid radical in question and the active compound. The patents in question contain a long list of substituents, but they do not specifically mention in the list the case in which W represents a saturated or unsaturated fatty acid radical, i.e. the patents in question do not mention BAFAC, Moreover, among the targeted effects of the compounds in question, there is not even the slightest hint that any of the compounds in question could be used to dissolve the cholesterol content of bile, prevent the formation of cholesterol-based gallstones, dissolve existing cholesterol-based gallstones, or reduce or prevent atherosclerosis. US-A-4,439,386 describes 7-acyl-chenodeoxycholic acid derivatives in which the acyl radical is derived from a saturated or unsaturated linear carboxylic acid having 3 to 18 carbon atoms; these compounds are useful in the treatment of gallstones. It describes, among others, ayl groups derived from butyric acid, caprylic acid, lauric acid, palmitic acid, stearic acid, oleic acid, linoleic acid and arachidonic acid. US-A-4,440,888 describes T-acyl-chenodeoxycholic acid derivatives in which the carboxylic acid radical is derived from a saturated or unsaturated linear carboxylic acid having 3 to 18 carbon atoms; these compounds are useful for the treatment of gallstones. It describes acyl groups derived from butyric acid, caprylic acid, lauric acid, palmitic acid, stearic acid, oleic acid, linoleic acid and arachidonic acid, among others. In this document, bile acid derivatives containing two carboxylic acid carboxylic acid groups are described. D.Krichevsky and colleagues; (Növel Derívatlves of 3.alpha.,7.alpba.-Dihydroxy-5.beta,-ohoian-24-oíc-Acid (Chenodeoxocholíc Áoid) and 3.aípha.,7.beta,-cholan~24-oio Acíd (Ursodeoxooholic acid)>!Steroids: Structure, Funotion, and Regulation, vol. 47, No. 1, pp. 41-48. ,, 7~acyl~eheno- and ursodeoxycholates, as well as methyl 3s7~diacyl~cheno~ and urseodeoxycholates are described. The acyl groups are derived from butyric acid, caprylic acid, palmitic acid, oleic acid, and linoleic acid. M. Kelsey et al., The Identification of Microbial Metabolites of Sulfolithocholic Acid Journal of Lipid Research, vol. 21, no. 6, 1980, pp. 761-759, discloses isothiocholic acid (ϋ_Α)3-β- fatty acid derivatives, i.e., palmitoyl, palmitoleyl, stearyl, and oleyl esters of ILA. M. Kelsey et al., Characterization of Microbial Metabolites of Sulfolithocholic Acid by High Performance Liquid Chromatography, vol. 14, no. 2, 1981, pp. 205-211, describe 3βpalmitoyl-5β-cholan-24-carboxylic acid. We have found that bile acids or their salts (BAFAC) linked to fatty acids (saturated or unsaturated) via an X-linkage bond can act as carriers for the transport of fatty acids into the bile, via the highly efficient intrahepatic circulation of bile acids and their salts. The esterification between the fatty acid(s) and the bile acid is not appropriate because it would be degraded by digestive juices and intestinal bacteria during absorption and intrahepatic circulation. Only a portion of the intact BAFAC remains in the bile. It has also been shown that BAFAC derivatives are absorbed from the intestine, transported to the liver and excreted in the bile. Said BAFAC derivatives improve the dissolution of cholesterol in bile and significantly delay its crystallization. Said BAFAC derivatives are therefore useful agents for preventing the formation or re-formation of cholesterol-based gallstones and for dissolving cholesterol-based gallstones. Administration of BAFAC also inhibits the crystallization of cholesterol in the vascular network. Under physiological conditions, digested bile acids or salts are absorbed into the intestine, enter the liver via the portal vein, and are excreted into the intestine via the bile. In this way, they participate in the intrahepatic circulation, and only a small amount reaches the blood circulation of the whole body (the vascular system). BAFAC derivatives behave more like lipids, which, after absorption in the intestine, enter the blood circulation via the lymphatic fluid. It has been shown that BAFAC derivatives are transported by both the lymphatic system and the portal vein. They enter the liver via both routes and are excreted into the bile. In each intrahepatic circulation, they reach the intestine, are partially reabsorbed again through the lymphatic system, and re-enter the vascular system before reaching the liver.Since the intrahepatic circulation has 10–12 cycles per day, the net effect of the process is the return flow of BAFAC into the vasculature. This effect is enhanced when the BÁFAC derivative is administered orally in several divided doses throughout the day. The cholesterol crystallization inhibitory effect of BÁFAC and its efficacy in dissolving existing cholesterol crystals have been proven. In this way, their value in reducing and / or preventing cholesterol crystallization in the vascular system, i.e. their efficacy in atherosclerosis, has also been proven. The present invention thus relates to a fatty acid-linked bile acid or bile salt of general formula (II): W - X - G in which formula G represents a bile acid or bile salt radical, W represents one or two fatty acid radicals having 8-28 carbon atoms, X represents a H-H bond between the bile acid or bile radical in question and the fatty acid radical(s). Preferred such compounds are mentioned in the appended claims. Suitable bile acids include, for example, cholanoic acid, chenodeoxycholanoic acid, ursodeoxycholanoic acid and deoxycholanoic acid. The bile acid used may be free or, as in bile, linked to glycine, taurine or other suitable amino acids. These possibilities are included in the definition of bile acid and thus also within the scope of the present invention. The coupling with the fatty acid radical is, depending on the bile acid used, mostly carried out at the 3-position of the backbone. The coupling with the fatty acid radical can be carried out at various positions, for example at the 6-, 7-, 12- and 24-positions. If the bile acid is linked to glycine or taurine, the coupling with the fatty acid radical cannot be carried out at the 24-position. The coupling with the fatty acid radical can be in the α or β configuration. *r Preferred fatty acids are saturated fatty acids, suitably having 14 to 22 carbon atoms. Preferred saturated fatty acids are behenic acid, arachidic acid, stearic acid, palmitic acid and myristic acid. When W represents two fatty acids, they are suitably linked at the 3- and 7-positions. The present invention also provides a pharmaceutical composition that allows the dissolution of cholesterol-based gallstones in bile and the prevention of their formation; and allows the prevention and / or reduction of atherosclerosis, the active ingredient of which pharmaceutical composition is a bile acid-fatty acid derivative of general formula (II). The pharmaceutical composition in question may be in the form of a tablet, capsule, solution, emulsion, etc. The pharmaceutical composition in question may contain additional compounds such as carriers, solvents, emulsifiers, absorption enhancers, substances inhibiting the synthesis of cholesterol or its secretion into the bile, etc. The pharmaceutical composition in question should preferably contain 0.1 - 1.5 g of active ingredient. The medicinal product should be taken once daily, preferably before bedtime. It can also be taken in several doses distributed throughout the day. The present invention also relates to the use of the bile acid-fatty acid derivative of general formula (II) or a pharmaceutical composition containing it for the production of medicaments suitable for dissolving cholesterol-based gallstones in bile and preventing their formation. The present invention also relates to the use of a bile acid-sulfur derivative of the general formula (II) or a pharmaceutical composition containing it for the preparation of medicaments suitable for preventing and / or reducing atherosclerosis. Cholesterol-based gallstones can be dissolved in bile and their formation can be prevented by administering the bile acid-fatty acid derivative of general formula (II) or a pharmaceutical composition containing it, The present invention also provides a method for preventing and / or reducing atherosclerosis by administering the bile acid derivative of formula (H) or a pharmaceutical composition containing the same. The present invention is illustrated in the following examples and figures without limiting the scope of the patent, The figures in the patent description: Figure 1A shows the coupling of colanic acid at the C-3 position with: behenic acid (C-22), arachidonic acid (C-20) and stearic acid (C-8); Figure 18 illustrates the preparation of stearoyl cholate coupled with glycine; Figure 1C shows the coupling of oleoyl cholate: Figure W shows the coupling of ursodeoxycholic acid with two molecules of stearic acid at positions 3 and 7 of the bile acid skeleton; Figure 2 shows the mass of cholesterol crystals in a model bile solution. Effects of BAFAC derivatives consisting of colanic acid linked to stearic (18C) and arachidyl (20C) acids (at the C-3 position). The compounds tested were used to replace 20 mol% of the sodium taurine cholate in the reference solution. Figure 3 shows the crystal formation time in model bile solution. Effects of the compounds shown in Figure 2. Figure 4 shows the cholesterol crystal mass in enriched human bile after 22 days of culture. The effect of 5 mM stearoyl(C18)cholate and arachidyl(C-20)-cholate added to the bile, compared to control bile and bile containing 5 mM cholanic acid, Figure 5 shows the crystal-center formation time in model bile, the effect of replacing 20 mol% sodium taurocholate with equivalent amounts of BAF derivatives, including stearoyl(C-18)-cholate, arachidyl(C-20)-cholate and distearoyl-ursodecylcholate, compared to model bile with or without replacing 20% ​​of HaTC with cholic acid; and Figures 8A and 6B show the concentration of stearoyl (C-18) cholate in hamsters 1, 2 and 3 hours after administration of 30 mg of compound. The concentrations are in blood taken from the heart, blood taken from the portal vein and bile from the gallbladder. Example I -amido~7a J2aS5~ko| (a) 1.15 g of 3β-behenyl amido-7α,12α-dihydroxy-5β-cholane-24-carboxylic acid methyl ester (1A, Figure i) (French Patent Application No. 1017756, December 18, 1952, Chem. Absir, 52:1293c) are dissolved in 30 ml of anhydrous dimethylformamide and 15 ml of triethylamine are added with stirring. To the resulting solution is added dropwise 1.13 g of behenoyl chloride dissolved in 10 ml of dimethylformamide and stirring is continued overnight >** * ;0. The reaction mixture is poured into water and extracted with methanol, the organic phase is then dried over sodium sulfate, evaporated to dryness and purified on silica gel with ethyl acetate and hexane (8:4 and 8:2} chromatographed with a mixture of 0.8 g of 3β-behenylamido-α,12α-dihydroxy-5β-cholan-24-carboxylic acid methyl ester (Figure 1A 2). 1H NMR spectrum (CDCIS, 3} ppm: 0.69 (s, CH3-18); 0.88 (t, 3 = 1 Hz, CH.3-23): 0.95 (s, CH3-19); 0.99 (d, 3=3 Hz, CH3-21j; 1.25: 1.14 [(s, CH2)20]; 2.14 (t, J=5 Hz, CR3~behenii);3.67 <scooch3>: 3.91 (d, J" 1.5 Hz, CR-7); 3.69 (s, 3=4 Hz, CH-12); 3.99 (m, CH-3); 5.60 (d, 3=4.5 Hz, -CH2CO-). (b) Dissolve 0.45 g of the methyl ester of the compound in 20 ml of methanol, add 2 ml of 1N sodium hydroxide and leave at room temperature for 24 hours. The methanol is then distilled off, 10 ml of water is added and the reaction mixture is extracted with ethyl acetate. The aqueous phase is then acidified with dilute hydrochloric acid to give a white precipitate which is washed with water to give 0.41 g of pure 3βbehenyl·amldo~7α,12α-dihydroxyl·5β-cholan~24"carboxylic acid (Figure 1A 3). Example 11 35-Ar3hidilamide7a<12«"dihydroxy-56-cholan-24-carbenoic acid (1 A~ (a) 1.0 g of 3p-amino~7a,12a-dihydroxy-5,8-cholan-24-carboxylic acid ethyl ester (1A. Figure 1} (see Example I]) is dissolved in 30 ml of anhydrous dimethylformamide, and 15 ml of triethylamine is added while stirring. To the resulting solution is added dropwise 1.0 g of arachidoyl chloride dissolved in 10 ml of dimethylformamide, and stirring is continued overnight. The reaction mixture is poured into water and extracted with methylene chloride, the organic portion is then sodium ίΦίΐ *λ*>: « « « * »»» * * dried over sulfate, evaporated to dryness and chromatographed on silica gel with a mixture of ethyl acetate and hexane (6:4 and 8:2) to give 0.6 g of 3p-arahidH-amyldo-7a, 12a-dihydroxy-5p-cholane-24-carboxylic acid methyl ester (Figure 1A 4). 1 H NMR spectrum (CDCIS, δ) ppm: 0.70 (s, CH3-18); 0.88 (t, 4=6 Hz, CB3~23); 0.96 (s, CH3-19): 0.99 <d, 4=3 Hz, CH3~21) 1,25; 1,14 j(s, CH2)ÍSj: 2,14 (I, 4=5 Hz, CH3~arahidíl}; 3,67 (s~ COOCHs); 3,91 (d, 4=1,5 Hz, CH-7); 3,96 (t, 4=4 Hz, CH-12); 4.4 (m, CH-3); 5.80 (d, 4=4.5 Hz, -CH 2 CONH). (b) 0.5 g of 3β-arachidamide-7α,12α-dihydroxy-5β-cholane-24-carboxylic acid methyl ester (Figure 1A 4) was dissolved in 20 ml of methanol, 2 ml of 1N sodium hydroxide was added, and the mixture was left to stand at room temperature for 24 hours. The methanol was then de-esterified, 10 ml of water was added, and the reaction mixture was extracted with ethyl acetate. The aqueous phase was then acidified with dilute hydrogen chloride, resulting in a white precipitate, which was washed with water to give 0.7 g of pure 3β-θΓ3ΚΙόίΙ"3ηί0ο-7α,12α-ΟΐΚί6ΓθΧί-5β-Κοταν-24-carboxylic acid (Figure 1A 5). 3ö~Sfeani-amide-7cí.12 <x~dihidröXl· Method 1 (a) 1.15 g of 3β-amino-7α, 12α-dihydroxy-5β-cholan-24-carboxylic acid methyl ester (Figure 1A 1) [see Example 1] was dissolved in 30 ml of anhydrous dimethylformamide and 15 ml of triethylamine was added while stirring. To the resulting solution was added dropwise 1.13 g of stearoyl chloride dissolved in 10 ml of dimethylformamide and stirring was continued overnight. The reaction mixture was poured into water and extracted with methylene chloride, then the organic «««« ♦·*** part was dried over sodium sulfate, evaporated to dryness and chromatographed on silica gel with ethyl acetate and hexane (6:4 and 8:2} to give 0.88 g of 3p-stearyl-amyldo-7a,12a-Oί0Ι8Γθχΐ-5β-ΚοΙόη~24~Η3Γ0οη88νηο10-ό5ζ1βΓί (Fig. 1A 6),1H NMR spectrum (CDCl3)8) ppm: 0.69 (s, CH3-18); 0.88 (t, 5=1 Hz, CH3-23); 0.95 (s, CH3-19); 0.99 (t, 5=3 Hz, CH3-21), 1.25; 1.44 [(s, 2.14 (t, 5=5 Hz, CH3-steaR); 3.67 (s~ COOCHs); 3.91 (d, 3=1.5 Hz, CH-7); 3.99 (m, CH-3); 4.4 (m, CH-3); 5.60 (d, 5=4.5 Hz, -CH3CONH), (b) 0.45 g of 3β-8Ζΐ©3τΗ~8πϋ0ο~7α,12α-άΙΐΉ<ΐΓθχί~5β~1<οΙάη-24~ -carboxylic acid methyl ester (Figure 1A-6) is dissolved in 20 ml of methanol, 2 ml of 1N sodium hydroxide is added, and the mixture is left at room temperature for 24 hours. Then, 10 ml of water is added, and the with ethyl acetate. Then the aqueous phase is acidified with dilute hydrochloric acid, resulting in a white precipitate, which is washed with water to give 0.41 g of 3 p-stearyl-amido-7a, 12a"dihydroxy-5p-cholan-24-carboxylic acid (1Ά, Figure 7), Melting point: 83-85 °C. 2.5 g of 3-amino-7a,12o-dihydroxy-5p-cholan-24-carboxylic acid (prepared according to Kramer et al., 5. of Lipid Research 24, 910, 1983) were dissolved in acetonitrile and added with stirring to a solution of 1.2 g of stearic acid and 3.8 g of N-hydroxysuccinimide in the same solvent. After 8 hours, the precipitate was filtered, washed with solvent and evaporated to dryness. The residue was added to a solution of 1.2 g of stearic acid in 10 ml of a 1:3 mixture of N-methylmorpholine and N,N'dimethylformamide. After being kept at room temperature overnight, the solution was washed with water « * ♦ * ♦ ·*« ♦♦♦ * * Dilute Φ * Φ * **< Φ*« * '♦, extract with ethyl acetate, thus obtaining 0.6 g of acid (Figure 1A-7), which is identical to the compound in Method 1. Method 3. g of stearic acid chloride solution was added to a toluene solution of 1.8 g of the amine (Fig. 18) at 0°C and allowed to stand at this temperature for one hour. The resulting solution was heated to 50°C for a further hour, acidified with 3N hydrochloric acid and concentrated. The solid was then washed with water and dried at 45°C to give the acid (Fig. 1A 7), which was identical to that described above. 24-amide (Figure 1B-17) (a) 0.5 g of 3β-stearyl amido-7α12α-dihydroxy-5β-colanic acid (Figure 1A 7) was dissolved in 25 ml of anhydrous 1,4-dioxane and cooled to -10°C. To the solution was added 0.5 ml of triethylamine and then 0.085 ml of chlorophyll-a, followed by stirring at the same temperature for 15 minutes. The solution was then allowed to warm to room temperature, 0.1 ml of triethylamine and 14 g of ethylglycine hydrochloride were added and allowed to stand overnight. The reaction mixture was poured into water, extracted with ethyl acetate and washed with water. The shaken portion was evaporated to dryness and chromatographed on silica gel with a mixture of ethyl acetate and hexane (60:40), pure ethyl acetate, and then ethyl acetate and methanol (0:1), yielding 0.27 g of product (Figure 1B-18). (b) 0.27 g of the above product was dissolved in 20 ml of methanol and 2 ml of 1N sodium hydroxide were added. After 24 hours, the methanol was evaporated to dryness, dissolved in water and extracted with ethyl acetate. The aqueous phase was washed with 1N hydrogen chloride. s. <x « * . *· Φ*χ· *Κ* * φ ν < ν'» $ * *Λ* is acidified. The resulting precipitate is washed with water and dried, yielding 0.24 g of dry material (Figure 18-17). Example V 38-Oleyl-amido-7a,12o-dihydroxy-5p-cholane-24-carboxylic acid (IC. Figure 20) (a) 1.6 g of 3p-amino-7a,12o-dihydroxy-5p-cholane-24-carboxylic acid methyl ester (1A, Figure 1) was dissolved in 30 ml of anhydrous dimethylformamide and 3 ml of trimethylamine was added with stirring. A solution of 1.38 g of oleyl chloride in 10 ml of anhydrous dimethylformamide was added dropwise and the resulting solution was left to stand overnight at room temperature. The reaction mixture was poured into water, extracted with ethyl acetate, and the organic portion was purified by washing with dilute hydrochloric acid, sodium hydrogen carbonate, and then with water. Evaporation to dryness under reduced pressure gives 3.1 g of material, which is chromatographed on silica gel with ethyl acetate / hexane (4:6 and 10:8) as the eluent, thus giving 1.8 o methyl ester (IC. Figure 19). (b) To a solution of 1.2 g of methyl ester in 20 ml of methanol at room temperature, 5 ml of 1N sodium hydroxide solution were added and the mixture was left to stand at room temperature for 48 h, then evaporated to dryness. The residue was dissolved in 20 ml of water and extracted with three 25 ml portions of ethyl acetate. The aqueous phase was acidified with hydrochloric acid to give a precipitate which was filtered. This residue was chromatographed on silica gel using ethyl acetate:hexane:acetic acid (10:4:0.3) as the eluent to give 0.3 g of 3p-oleyl amido-7a,12a-dihydroxy'5p-cholan-24-carboxylic acid (IC. Figure 20). Example VI - ~ ©« $ φ * 'ν'· £ * Φ * ν Φ * XV Α * .♦·'© ·»*« * 3β,7α-8ΐ5ζΙοοηΙ-3ΓηΙάο>5&~αΓζο0βοχΙ·~&οΐ3η~24~^3^οπ53Υ <10. (a) 20 g of ΡΓ2θ0οοχί~Κοίόη~24^ΡΓ0οη$3νρί were dissolved in 2ÖÖ ml of absolute methanol, 1 ml of concentrated sulfuric acid was added, and the mixture was stirred for 24 hours. Most of the solvent was distilled off, and the residue was poured into water and then extracted with methylene chloride. The organic phase was washed with sodium hydrogen carbonate and sodium chloride solution, and then evaporated to dryness to give 19.5 g of 3α,7β~ άΙΜ0ΓθχΙ~5β-ηΓζοόβοχΙ~ΚοΙόη~24-Κ:3^οη83¥ methyl ester (1 D~ NMR spectrum (CDCl ppm: 0.68 (s, CH-3~1 3=1 Hz, CH3~23); 0.93 (s, CH3-19); 0.94 (d, 3=3 Hz, CH3~21); 3.58 <m, CH-3, CH-7): 3,65 (s-COOCH3). (b) 4.06 g of methyl ester (Figure 1D 21) was dissolved in 30 ml of anhydrous pyridine and cooled to 0 °C. A solution of 1.49 g of methanesulfonyl chloride in 5 ml of pyridine was added to the reaction mixture while stirring. After standing at the same temperature for 3 hours, the reaction mixture was poured into ice water and extracted with ethyl acetate. The organic portion was washed with hydrochloric acid, sodium hydroxide and sodium chloride, filtered and concentrated under reduced pressure. The residue consisting of four compounds was chromatographed on a silica gel column using a mixture of ethyl acetate and hexane as the solvent. The 5.3 g less polar compound is the desired 3α,7β-dimezyl-5β-ursodeoxycholan-24carboxylic acid methyl ester (Figure 1D 22). 1 H NMR spectrum (€DCi 3 , 5) ppm: 0.65 (s, CH 3 -18); 0.90 (d, J=4 Hz, CH3~23); 0.97 (s, CH3-19); 1.2 (t, 3=3 Hz, CH3-21); 2.97 (s, CH3.SO2); 2.98 (s, CH 3 SO 2 ); 3.84 (s, CH 3 SO 2 ); 4.09 (g, 3=12 Hz, H-7); 4.82 (m, H-7). ** # ♦*« < RS (c) Dissolve 5.65 g of άίΓηθζίΙ-5Ζ8ΓΗΐ8ζβ1ζ in 50 ml of anhydrous dimethylformamide, react with anhydrous sodium azide, and then heat at 130 C for 2 hours. Cool the reaction mixture, pour it into ice water, and extract with ethyl acetate. The shaken portion is then washed with sodium acetate and sodium chloride solutions, filtered, and evaporated to dryness, thus obtaining 4.5 g of 3β,7α-diazido-5β-ursodeoxycholine-24-carboxylic acid methyl ester (d) 4.5 g of the diazido compound (Figure 10-23) are dissolved in 120 ml of methanol, 150 mg of 5% palladium on carbon are added, and hydrogenation is carried out at atmospheric pressure for 4 days. The hydrogenation is repeated with another 150 mg of 5% palladium on carbon. The hydrogenated mixture was filtered and evaporated under reduced pressure to obtain 3 g of 33,7α-dla-amino-5-pyrodeoxycholic acid-24-carboxylic acid methyl ester (Figure 10, 24). 1 H NMR spectrum (CDCl 3 ) ppm: 0.65 (s, CH 3 -18 ); 0.92 (d, J~4 Hz, CH3-23); 0.96 (s, CH3-19); 1.2 (t, J-3 Hz, CH3-21); 3.68 (S-COOCH 3 ); 3.72: 3.95 (m, 2H~7.3). (e) 1.47 g of 3ps7a-diamino-5p-ursodeoxycholan-24-carboxylic acid methyl ester (Figure 10 24) was dissolved in 50 ml of a 1:1 mixture of anhydrous DMSO and DMF, 2 ml of triethylamine and 30 mg of dimethylaminopyridine and 5.1 g of stearic anhydride were added. The reaction mixture was heated to 50 C, stirred for 18 hours, poured into ice water and extracted three times with ethyl acetate. The organic phase was washed with hydrochloric acid, sodium bicarbonate and sodium chloride solution. After evaporation of the organic solvent, 2.05 g of an oily residue were obtained. The residue was extracted on silica gel, ethyl acetate : hexane «φ«* Φ V «φ Φ * ♦ « Separation of X Φ φ * ♦ ** ♦» * * ·» φ » * ♦ (1:4) with solvent yields several fractions, one of which, according to its mass spectrum and 1H NMR spectrum, is 80 mg of the desired 3β,7α~άίδζΙβ3ΓΗ~3Πΐί80-5β-θΓζο4οχί-ΚοΜη-24-Κ3Γ~ bonic acid methyl ester (1D, Figure 25). Mass spectrum FAB: MH+937 (MW) 938, 1H NMR spectrum (CDCl3, δ) ppm: 0.86 (s, CH3-18); 0.88 (d, 3=4 Hz, CH3-23): 0.98 (s, CH3-19); 1.2 (t, 3=3 Hz, CH3~21); 1.26 [s, <CH2)t6J; 3,84 (s, COOCK3); 3,05 (d, 3=7 Hz, H~7); 5,75 (m, H-3). (f) 78 mg of the methyl ester (Figure 1D-25) was dissolved in 20 ml of methanol, 2 ml of 1N sodium hydroxide was added and the mixture was left to stand at room temperature for 43 hours. The methanol was evaporated under reduced pressure, the residue was dissolved in 25 ml of water, filtered and then acidified with dilute hydrochloric acid to obtain a precipitate, which was 3β,7α-distearyl-amido-5β-ursodeoxycholan-24-carboxylic acid (Figure 1D-28). Cholesterol (Sigma, St. Louis, Mo.) was recrystallized twice from hot ethanol; Na-taurocholate (Na-TC: Sigma, St. Louis, Mo.) was recrystallized twice from ethanol and ether (3, L Popé, 3. Lipid Res. 3, (1967) 146-147); egg white lecithin (EYL) (Avanti Polar Lipids, Alabaster, ÁL) was used without further purification. All lipids used in our studies were pure, as compared by thin-layer chromatography. 1. Preparation of bile samples A mixture of egg white lecithin, cholesterol and Na-laurocholate was dissolved in a 2:1 volume chloroform / methanol mixture and dried at room temperature with nitrogen, lyophilized overnight and stored at -20°C under argon until use. Model bile solutions were prepared by suspending the dried bile in a solution containing 150 mM sodium chloride, 1.5 mM disodium EDTA, 50 mM Tris-HCl (pH 8.0) and incubating the suspension at 55°C for 1 h. The solubilized model biles were incubated at 37°C in a closed tube under argon for the duration of the experiment. Samples of the model solutions were taken daily. All model solutions are prepared in triplicate and treated under the same conditions throughout the experiments. The composition of the model bile: 15 mM cholesterol, 30 mM egg white lecithin and 150 mM Na-lauro-cholate. The further model bile solutions tested are prepared by replacing egg white lecithin or Na-taurocholate by 10~20%~of the synthetic bile acid conjugate. 8. Natural bile Natural human gallbladder bile is derived from bile from patients who have undergone surgery for cholesterol-based gallstones. Pooled bile from multiple patients is enriched in cholesterol to promote crystallization by either coating it with dried cholesterol or mixing it with concentrated model bile before use in experiments. * * > »♦<4 ««> 2. Evaluation of cholesterol crystal formation and growth 2.1 Crystal detection time (COT) tests The COT value (also known as crystal formation time) was determined according to the report of Holan et al. in Gasfroenterology 77, (1979) 811-817. Samples taken from each model bile were examined daily with a polarized light microscope. The COT was determined as the time when at least three crystals first appeared in the field of view of the microscope at 100x magnification. 2.2 Determination of crystal growth rate (CGR) Crystal growth is monitored spectrophotometrically using a microplate reader (SPECTRA-STL, Austria) (GJ Somjen et al., d. Lipid Res. 38, (1977) 1048 - 1052). 50 μΙ of the Ifpid solutions are mixed with an equivalent amount (200 mM) of Na-taurodeoxycholate in the wells of the microplate by vigorous shaking. After being kept at room temperature for 80 minutes, the microplates are shaken again and the absorbance of each well is read at 405 nm. Each model is prepared in triplicate, measurements are made on two parallel samples. Data collection and analysis are performed using an IBM compatible personal computer, and the average optical density (OD) of triplicate samples is calculated, and the average OD changes for each solution are plotted on a curve. The slope in the steepest region of the curve is determined by fitting a linear regression line calculated from at least three measurements, this is the CGR value to be determined. For each model, we calculate the differences in CGR and OD between days 0 and 14. 2.3 Crystal mass measurement On the last day of the experiment (day 14), each sample was subjected to a chemical analysis of cholesterol, as previously described (GJ Somjen, see above). The samples were collected from the microwells and centrifuged at 70,000 rpm for 5 minutes in an Airfuge (Beokman) centrifuge. Separate determinations were made for the whole sample (centrifugation time) and for the supernatant solution. The amount of cholesterol in the precipitated dry matter was calculated by subtracting the amount in the supernatant solution from the total amount. The crystalline nature of the dry matter was confirmed by polarized light microscopy. The mass of the crystalline material, as the difference between days 0 and 14 of incubation, was also measured spectrophotometrically. 3. Data analysis Each lipid dispersion was prepared in triplicate and two replicate measurements were performed for each sample. The mean OD values ​​and standard errors were calculated. Crystal growth rates were calculated from the crystal growth curves using linear regression analysis as described above. Comparisons between different model solutions were performed using one-way analysis of variance. The composition of the bile solution is as follows: mM cholesterol, 30 ml egg white leolin, 150 ml Na-tauro-cholate. Prepare according to Example VIL. In the test solutions, 20 mol% of Na-tauro-cholate is replaced by an equal amount of each of the fatty acid / bile acid conjugates tested. The results obtained with the conjugates of C18 and C20 saturated fatty acids linked to colanic acid at the 3-position are shown in Figures 2 and 3. Figure 2 shows the effect of these conjugates on the cholesterol crystal weight after 14 days of incubation in the reference and test solutions. All of the above conjugates reduced the final crystal weight compared to the reference solution. The 18-carbon conjugate reduced the crystal weight to 14% of the reference solution value, and the 20-carbon conjugate reduced it to 38%. In another experiment, the 22-carbon conjugate showed similar activity to the 20-carbon conjugate. Figure 3 shows the crystal formation time (crystal detection time) for the different test solutions compared to the reference solutions. When 20% of the Na-taurocholate is replaced by the indicated conjugates, the crystal formation time is prolonged for the C and C conjugates. The 20-carbon conjugate prolonged the crystal formation time by more than 360%. Example SX The bile from human gallbladders obtained during cholecystectomy is combined and enriched with concentrated lipids to enhance cholesterol crystallization. The final concentration of added lipids in the bile is: 60 mM Na-taurocholate 18.4 mM egg white lecithin and 9.2 mM cholesterol. The cholesterol crystals are removed from the enriched bile by ultracentrifugation at 5000 rpm for 1 hour and then divided into four tubes. The first tube contains only enriched bile (comparative). The following 5 μM solutions are added to the other three tubes: cholanic acid, 18-carbon siaroyl cholate and 20-carbon arachidyl cholate. After 22 days of incubation at 37°C, the bile was centrifuged in an airfuge at 70,000 rpm for 5 minutes. The sediment was removed and the cholesterol content was measured chemically. The results are shown in Figure 4 as the amount of cholesterol in the weight of the precipitated crystal, in pmol.It is clear that bile acid / fatty acid conjugates significantly reduce cholesterol crystallization compared to control bile prepared with or without colanic acid. Example X As a reference solution, a model bile solution is prepared as described in Example VII, with the same lipid composition. In all other samples, 20 mol% Na-taurocholate is replaced by: equivalent amounts of colanic acid, 18-carbon cholate, and 20-carbon cholate (all saturated fatty acids attached at the 3-position of colanic acid), and distearoyl ursodeoxycholate (in which the stearic acid radicals are attached at the Οπέ and 7-positions of the bile acid, in equal proportions). All samples were incubated at 37°C as described in Example VII and the time of crystal formation was determined periodically by light microscopy. The results are shown in Figure 5. The results demonstrate that: ->»«·♦ «««* ««*« « * Λ » *** χ « ♦ * * < ♦ .»» ♦»* * * 1) All conjugates tested (BAFAC) delayed cholesterol crystallization compared to the comparative model bile and an equivalent amount of colanic acid. 2) BAFAC derivatives containing longer chain fatty acids are more effective than those with shorter chains. 3) The conjugate containing two fatty acids (distearol ursodeoxycholate) is particularly effective. - 180 g female hamsters are given 30 mg of 18-carbon cola via a gastric tube. Each animal is euthanized 1, 2 and 3 hours after administration. Blood samples are taken from the heart and portal vein, as well as bile from the gallbladder. Two groups of animals are examined in parallel (A and B). Stearoyl cholate levels are measured using a high-pressure liquid chromatography device (Kontron Switzerland) with a 206 nm UV detector. The results are shown in Figures 6A and 8B. In group A: the concentrations in blood samples taken from the heart after 1, 2 and 3 hours were 99, 7, 2 μΜ, while the blood samples from the portal vein were 68, 99 and 133 μΜ. The concentration of 18-carbon cholate in the gallbladder was 548 and 270 μΜ, after 2 and 3 hours. Similar results were obtained in group B. The data shows that: 1) 18-carbon stearoyl cholate is absorbed from the intestine. 2) It is transported throughout the body (through the lymphatic system) and through the portal vein, 3) The compound is actively secreted into the bile and becomes concentrated there. As a reference solution, a model bile solution is prepared using the procedure described in Example VII with the same lipid composition. Colanic acid, stearoyl (C-18:0) cholate and oleoyl (C-18:1) cholate at 20 mM were added to the test solutions. All samples were incubated at 37°C for 100 hours. The difference in optical density between 0 and 100 hours was used to measure the total crystal weight at 100 hours (as described in Example VII). Compared to the reference solution (100%), the crystal weight was 114% for colanic acid, 82% for stearoyl cholate and 55% for oleoyl cholate. These results demonstrate that both saturated and unsaturated (oleic) acid-containing BAFAO derivatives reduce cholesterol crystallization compared to the control bile and an equivalent amount of colanic acid.

Claims

Patent claims 1. (II) A bile acid or bile salt-fatty acid conjugate of the general formula W - X - G in which G represents a bile acid or bile salt radical, W represents one or two fatty acid radicals having 6-26 carbon atoms, X represents an NH linking group between the bile acid or bile salt radical in question and the fatty acid radical(s).

2. The bile acid or bile salt-fatty acid conjugate according to claim 1, characterized in that the bile acid is cholanoic acid, chenodeoxycholanoic acid, ursodeoxycholanoic acid or deoxycholanoic acid.

3. Bile acid or bile fatty acid conjugate according to claim 1 or 2, characterized in that the bile acid is conjugated with glycine, taurine or a corresponding amino acid.

4. A bile acid or bile salt-fatty acid conjugate according to any one of claims 1-3, characterized in that the fatty acid radical is attached to the 3-position of the bile acid skeleton.

5. A bile acid or bile salt-fatty acid conjugate according to any one of claims 1-3, characterized in that the fatty acid radical is a member of the bile acid skeleton. It is attached to the 6-, 7-, 12-, or 24-position.

8. A bile acid or bile salt-fatty acid conjugate according to any one of claims 1-5, characterized in that the linkage between the fatty acid radical and the bile acid is in the α or β configuration.

7. The bile acid or bile salt-fatty acid conjugate of claim 1, wherein the saturated fatty acid has 14-22 carbon atoms.

8. The bile acid or bile salt-fatty acid conjugate according to any one of claims 1-7, characterized in that the saturated fatty acid is behenic acid, arachidic acid, stearic acid, palmitic acid or myristic acid.

9. 38~Behenylamido-7o,12a“dihydroxy~S8~cholan~24~carboxylic acid.

10. 38-Arachidylamido-7α,12α-dihydroxy-5β-cholane-24-carboxylic acid.

11. 38-Stearic-amyldo-7a(12a-dihydroxy-58-cholan-24-carboxylic acid.

12. 3β~ΡοΙίηΗοΠ^π·ηόο-7ο,12ο~όΙΜόηοχΙ~58~ΗοΙόη~24~Ι^όοηacid.

13. 38-bis(1-amino)-7o>12a-dihydroxy-S8-cotane-24-carboxylic acid, 14. N-(Carboxymethyl)-38-s-earyl-amido-7o,12€2-dihydroxy-58-cholan-24-amld.

15. A bile acid or bile salt-fatty acid conjugate according to any one of claims 1-4, 8 and 7, characterized in that W represents two fatty acid groups attached to the 3- and 7-positions of the fatty acid backbone, 16. A pharmaceutical composition which enables the dissolution of cholesterol-based gallstones in bile and the prevention of their formation, or which enables the prevention and / or reduction of atherosclerosis, the active ingredient of which pharmaceutical composition is a bile acid or bile salt fatty acid derivative of general formula (Ί1) according to any one of claims 1-15.

17. The pharmaceutical composition according to claim 16, wherein the pharmaceutical form is a tablet, capsule, solution or emulsion.

18. A pharmaceutical composition according to claim 18 or 17, which contains as additives a carrier, a solvent, an emulsifier, an absorption enhancer, or a substance that inhibits cholesterol synthesis or inhibits the secretion of cholesterol into the bile.

19. A pharmaceutical composition according to any one of claims 18-18, wherein the amount of active ingredient is between 0.1 and 1.5 g.

20. Use of a bile acid or bile salt-fatty acid conjugate according to any one of claims 1-15, or a pharmaceutical composition according to any one of claims 16-19, for the preparation of a medicament suitable for dissolving cholesterol-based gallstones in bile or preventing their formation.