Tetrazole derivatives of bile acids, a process for their production and their use as medicines.

The invention relates to bile acid derivatives of the formula I.         G1 - X - G2 I, Processes for their preparation, pharmaceutical preparations based on these compounds and the use of bile acid derivatives as medicaments.

The compounds according to the invention have a high affinity for the specific bile acid transport system of the small intestine and inhibit bile acid absorption in a concentration-dependent and competitive manner.

Bile acids have an important physiological function in fat digestion, e.g. B. as cofactors of pancreatic lipases and as natural detergents for solubilizing fats and fat-soluble vitamins. As the end product of cholestin metabolism, they are synthesized in the liver, stored in the gallbladder and released from it by contraction into the small intestine, where they exert their physiological effect. Most of the secreted bile acids are recovered via the enterohepatic cycle. They return to the liver via the mesenteric veins of the small intestine and the portal vein system. Both active and passive transport processes play a role in reabsorption in the intestine. The majority of the bile acids are reabsorbed at the end of the small intestine, the terminal ileum, by a specific Na⁺-dependent transport system and return to the liver with the mesenteric vein blood via the portal vein, so that they can be secreted again into the bile by the liver cells. In the enterohepatic cycle, the bile acids appear both as free acids and in the form of glycine and taurine conjugates.

Non-resorbable, insoluble, basic, cross-linked polymers have been used for some time to bind bile acids and are used therapeutically because of these properties. Bile acid derivatives described in patent application EP-A-0 489 423 have a high affinity for the intestinal bile acid transport system and therefore allow specific inhibition of the enterohepatic circulation. All diseases in which inhibition of bile acid absorption in the intestine, in particular in the small intestine, appears to be desirable, are regarded as therapy objects. For example, cholagenic diarrhea after ileum resection, or increased cholesterol blood levels, are treated in this way. In the case of high cholesterol blood levels, intervention in the enterohepatic circulation can lower this level. By lowering the bile acid pool in the enterohepatic circulation, the corresponding new synthesis of bile acids from cholesterol is forced in the liver. LDL cholesterol, which is found in the bloodstream, is used to cover cholesterol requirements in the liver, with the hepatic LDL receptors being increasingly used. The resulting acceleration of LDL catabolism has an effect by reducing the atherogenic cholesterol content in the blood.

Most natural bile acids have a terminal carboxyl group (C atom 24) in the side chain on the D ring of the steroid skeleton. The carboxyl group is in free form or conjugated with an amino acid.

The tetrazol-5-yl group is an isosteric group for the carboxyl function, ie it has steric, electronic and acidic properties similar to the carboxyl function itself. It is known from medicinal chemistry that when the carboxyl group of certain active ingredients is replaced by a tetrazol-5-yl radical the affinity for enzymes and proteins is maintained or increased and thus a better potency can be achieved (Drugs of the Future 1992, 17 (7) 575 to 593).

The task was to find new drugs that are able to reduce the atherogenic cholesterol in the blood or to influence the enterohepatic circulation with regard to increased bile acid excretion and the subsequent lowering of the cholesterol level.

This object is achieved by the tetrazole bile acid derivatives according to the invention.

The invention therefore relates to tetrazole bile acid derivatives of the formula I.         G1 - X - G2 I in the<dl id="dl0001"><dt>G1</dt><dd>H, a bile acid residue or a modified bile acid residue which is modified on the hydroxy functions and / or on the carboxyl group, and</dd><dt>G2</dt><dd>means a bile acid residue or a bile acid residue derivatized at the hydroxy functions and carrying a tetrazolyl residue in the side chain, and</dd><dt>X</dt><dd>represents a bridging group or a covalent bond, where G1 and G2 are connected as desired via X.</dd></dl>

The compounds according to the invention have a high affinity for the specific bile acid transport system of the small intestine and inhibit bile acid absorption in a concentration-dependent and competitive manner.

Furthermore, the compounds according to the invention are not themselves absorbed and therefore do not enter the bloodstream. By using this principle of action, the enterohepatic cycle of bile acid can be interrupted very specifically and efficiently.

By using the compounds according to the invention, it is possible to reduce the amount of bile acid in the enterohepatic circulation, so that the serum cholesterol level is reduced. Avitaminoses are unlikely to be used, as is the influence on the absorption of other drugs or a negative effect on the intestinal flora. Furthermore, the side effects known from the polymers (constipation, steatorrhea) are not observed, ie fat digestion is not adversely affected. Because of the high affinity for the specific bile acid transport system of the small intestine, low daily doses are sufficient, so that the acceptance of such drugs by doctors and patients will be very high.

Compounds of the formula I are preferred in which the linkage between the steroid structures of G1 and G2 does not take place via identical rings of the steroid structure, where X is a covalent bond or a bridging group,<dl id="dl0002"><dt>G1</dt><dd>H, a bile acid residue or a bile acid residue modified on the hydroxy functions and / or on the carboxyl group,</dd><dt>G2</dt><dd>means a bile acid residue or a bile acid residue derivatized on the hydroxy functions, which bears a tetrazolyl residue in the side chain.</dd></dl>

Particularly preferred are compounds of the formula I in which the linking of G1 and G2 takes place via the ring D of G1 and the ring A of G2, where G1 is H or a radical of the formula II,<chemistry id="chem0001" num="0001"><img file="EP0624596A2_D0001.tif" /></chemistry> in the<dl id="dl0003"><dt>R (1)</dt><dd>H, an alkyl radical or alkenyl radical with up to 10 carbon atoms, which is branched or unbranched, a cycloalkyl radical with 3 to 8 carbon atoms, a benzyl radical which is unsubstituted or substituted 1 to 3 times with F, Cl, Br, (C₁-C₄) alkyl or (C₁-C₄) alkoxy, a diphenylmethyl radical which is unsubstituted or substituted 1 to 3 times with F, Cl, Br, (C₁-C₄) alkyl or (C₁-C₄) alkoxy, a triphenylmethyl radical which is unsubstituted or substituted 1 to 3 times is with F, Cl, Br, (C₁-C₄) alkyl or (C₁-C₄) alkoxy, a C₁-C₄ alkoxymethyl or a tetrahydroxylamyl radical or a radical<chemistry id="chem0002" num="0002"><img file="EP0624596A2_D0002.tif" /></chemistry> where L H, an alkyl radical or alkenyl radical with up to 10 C atoms, which is branched or unbranched, a cycloalkyl radical with 3 to 8 C atoms, a phenyl radical which is unsubstituted or 1 to 3 times substituted with F, Cl, Br , (C₁-C₄) alkyl or (C₁-C₄) alkoxy, a benzyl radical which is unsubstituted or substituted 1 to 3 times with F, Cl, Br, (C₁-C₄) alkyl or (C₁-C₄ ) -Alkoxy means</dd><dt>X</dt><dd>is a single bond or a bridge member of the formula III,<chemistry id="chem0003" num="0003"><img file="EP0624596A2_D0003.tif" /></chemistry> in which A is an alkylene chain which is branched or unbranched and can optionally be interrupted by -O-, -S- or phenylene, the linkage on the phenylene ring being ortho, meta or para and the chain a total of 2 to 12, preferably 2 to 6 , Chain links p comprises, B is an alkylene chain which is branched or unbranched and can optionally be interrupted by -O-, -S- or phenylene, the linkage on the phenylene ring being ortho-, takes place meta or para and the chain comprises a total of 2 to 12, preferably 2 to 6, chain links n, L (1), L (2), L (3) are the same or different and have the meaning of L, and</dd><dt>q</dt><dd>0 until 5</dd><dt>r</dt><dd>0 or 1</dd><dt>s</dt><dd>0 or 1</dd><dt>t</dt><dd>0 or 1 mean</dd><dt>R (2) to R (5),</dt><dd>where R (2) and R (3) or R (4) and R (5) each together the oxygen of a carbonyl group or individually and in each case independently of one another H, -OL, -SL, -NHL, tetrahydropyranyloxy or C₁-C₄- Alkoxymethoxy mean, where L has the meaning given above and</dd><dt>G2</dt><dd>represents a radical of the general formula IV<chemistry id="chem0004" num="0004"><img file="EP0624596A2_D0004.tif" /></chemistry> where Z<chemistry id="chem0005" num="0005"><img file="EP0624596A2_D0005.tif" /></chemistry> with m = zero to 4, n = zero or 1 and o = zero to 4,</dd><dt>V</dt><dd>-O-,<chemistry id="chem0006" num="0006"><img file="EP0624596A2_D0006.tif" /></chemistry> -CH₂-, -CH₂CH₂-</dd><dt>W</dt><dd>H, or if V = -CH₂-, -CH₂CH₂-, also OH and R (6) to R (9) have the meaning given under R (2) to R (5).</dd></dl>

Compounds of the formula I in which G1 is H or a radical of the formula II are very particularly preferred<chemistry id="chem0007" num="0007"><img file="EP0624596A2_D0007.tif" /></chemistry> in which<dl id="dl0004"><dt>R (1)</dt><dd>H, formyl, acetyl, benzoyl, methoxymethyl or tetrahydropyranyl,</dd><dt>R (2) to R (5),</dt><dd>wherein R (2) and R (3) or R (4) and R (5) each together the oxygen of a carbonyl group or individually and independently of one another H, OH, O-formyl, O-acetyl, O-benzoyl, methoxymethoxy or Tetrahydropyranyloxy mean</dd><dt>X</dt><dd>represents a covalent bond or one of the following bridge groups<chemistry id="chem0008" num="0008"><img file="EP0624596A2_D0008.tif" /></chemistry></dd><dt>G2</dt><dd>means a radical of formula IV<chemistry id="chem0009" num="0009"><img file="EP0624596A2_D0009.tif" /></chemistry> in which</dd><dt>V</dt><dd>-O-,<chemistry id="chem0010" num="0010"><img file="EP0624596A2_D0010.tif" /></chemistry></dd><dt>W</dt><dd>H</dd><dt>Z.</dt><dd><chemistry id="chem0011" num="0011"><img file="EP0624596A2_D0011.tif" /></chemistry> with m = 1 to 3, n = zero or 1 and o zero, 1 or 2 R (6) to R (9) have the meaning given above under R (2) to R (5).</dd></dl>

The invention further relates to a process for the preparation of compounds of formula I, which is characterized in that<ul id="ul0001" list-style="none"><li>a) in the case of X = single bond, suitable reactive forms of G1 and G2 are reacted with one another by methods known in principle or</li><li>b) in the case of X = bridge group<ul id="ul0002" list-style="none"><li>α) reactive forms of G1-X with G2 or</li><li>β) reactive forms of G2-X with G1</li></ul> by reacting with one another in principle known processes.</li><li>a) X = single bond The G1 bile acids are used either in free form or in protected form. After the linkage with G2, which is also in free or protected form, the protective groups are split off if necessary. Formyl, acetyl or tetrahydropyranyl are expediently suitable as protective groups for the alcohol groups. The tetrazolyl function is either already present in G2 before the linkage or is introduced via a carboxyl group of G2 after the linkage with G1. For example, bile acid reacts preferentially at position 3, but also at position 7 with activated forms of carboxylic acids, such as acid chlorides or mixed anhydrides with the addition of bases such as trialkylamine, pyridine, but also NaOH at room temperature in suitable solvents such as tetrahydrofuran, methylene chloride or ethyl acetate, but also Dimethylformamide (DMF) or dimethoxyethane (DME). The various isomers can e.g. B. be separated chromatographically. The reaction can be carried out selectively by using suitable protective groups. Corresponding amino bile acids can be converted analogously into corresponding amides. Here too, the reaction can be carried out with either protected or free bile acids. Analogously, further compounds according to the invention can be linked using known standard methods.</li><li>b) X = bridge group The methods specified under a) are also used to link G1-X with G2 or G1 with X-G2. Expediently, the bile acid part is also used here either protected or unprotected. A preferred manufacturing process is to react reactive forms of G1 with reactive forms of X-G2. Otherwise, after the linkage, protective groups are split off and converted into a tetrazole derivative.</li></ul>

The preparation of tetrazolyl-bile acid building blocks or tetrazolyl-bile acid is described in the following formula schemes.<chemistry id="chem0012" num="0012"><img file="EP0624596A2_D0012.tif" /></chemistry> C-22-tetrazolyl-cholanic acid derivatives X are accessible by first shortening the side chain of a C-24-cholanic acid V by one carbon atom to the nitrile VI (J. Lip. Res. <u>29</u>, 1387, 1988). The protective groups are split off by saponification under mild conditions and compounds of type VII are obtained. These nitriles VII can now directly with trialkyltin azides in a suitable solvent, such as. B. toluene, at elevated temperatures to the tetrazole derivatives X are implemented. However, it may be advantageous to protect the free OH groups from the tetrazole formation step, e.g. B. with THP or MOM protective groups to compounds VIII. The reaction to the protected tetrazole derivative IX takes place under the conditions mentioned above. Removal of the protective groups in turn leads to compounds of type X.<chemistry id="chem0013" num="0013"><img file="EP0624596A2_D0013.tif" /></chemistry> To produce C-23 tetrazolylcholanoic acids XVI, iodine compounds of type XI, for example, are converted into nitriles XII by nucleophilic substitution with alkali metal cyanides. After the protective groups have been split off, the nitriles XIII can either be converted directly to the tetrazole compounds XVI or the possibility can be used via the protected derivatives XIV and XV.<chemistry id="chem0014" num="0014"><img file="EP0624596A2_D0014.tif" /></chemistry><chemistry id="chem0015" num="0015"><img file="EP0624596A2_D0015.tif" /></chemistry> C-24 tetrazole derivatives are obtained by protecting compounds of type XVII on the hydroxyl groups and on the acid function. The resulting ester functions of compounds of type XVIII are reduced to primary hydroxyl groups (XIX). The hydroxyl group is activated by a methane or toluenesulfonyl radical (XX) and substituted with an alkali metal cyanide; thereby type XXI compounds are obtained. The reaction of nitrile to tetrazolyl compounds (XXII) and the subsequent cleavage of the protective groups to XXIII is carried out according to the above-described method. An amino function can be produced from the free hydroxy function in position 3 or in the introduced link X analogously to the processes already described (EP-A-0 489 423) via compounds of the formulas XXIV and XXV and compounds of the type XXVI are obtained.<chemistry id="chem0016" num="0016"><img file="EP0624596A2_D0016.tif" /></chemistry> From natural bile acids or from modified bile acids, e.g. B. from dimeric derivatives, tetrazole derivatives can be prepared by reacting the free carboxyl group with 5-aminotetrazole after activation. When using the usual peptide coupling reagents, the yields in this reaction are low. It is therefore advisable to protect the free hydroxy functions of the bile acid derivative XXVII, e.g. B. with formyl or acetyl protective groups. From the protected compounds XXVIII with phosphorus pentachloride or thionyl chloride, for. B. in THF, which produces acid chloride. Reaction of the acid chloride with dry 5-aminotetrazole gives compounds of the type XXIX, from which the end products XXX are obtained by simply removing the protective groups.

The compounds have valuable pharmacological properties and are therefore particularly suitable as hypolipidemics.

The invention therefore also relates to medicaments based on the compounds of the formula (I) and the use of the compounds as medicaments, in particular for lowering the cholesterol level.

The compounds according to the invention were tested biologically by determining the inhibition of the [3 H] taurocholate uptake in brush border membrane vesicles of the rabbit ileum. The inhibition test was carried out as follows:

1. Preparation of brush border membrane vesicles from the ileum of rabbits

The preparation of brush border membrane particles from the intestinal cells of the small intestine was carried out using the so-called Mg²⁺ precipitation method. Male New Zealand rabbits (2 to 2.5 kg body weight) were injected intravenously with 0.5 ml of an aqueous solution of 2.5 mg of tetracaine HCl, 100 T 61^R and killed 25 mg of mebezonium iodide. The small intestine was removed and rinsed with ice-cold physiological saline. The terminal 7/10 of the small intestine (measured in the oral rectal direction, ie the terminal ileum, which contains the active Na⁺-dependent bile acid transport system) were used to prepare the brush border membrane vesicles. The intestines were frozen in plastic bags under nitrogen at -80 ° C. To prepare the membrane vesicles, the frozen intestines were thawed at 30 ° C in a water bath. The mucosa was scraped off and dissolved in 60 ml ice-cold 12 mM Tris / HCl buffer (pH 7.1) / 300 mM mannitol, 5 mM EGTA / 10 mg / l phenylmethylsulfonyl fluoride / 1 mg / l trypsin inhibitor v. Soybeans (32 U / mg) / 0.5 mg / l Trypsin Inhibitor v. Bovine lung (193 U / mg) / 5 mg / l bacitracin suspended. After dilution to 300 ml with ice-cold distilled water, an Ultraturrax (18-rod, IKA Werk Staufen, FRG) was used for 3 minutes at 75% max. Performance homogenized under ice cooling. After adding 3 ml of 1 M MgCl₂ solution (final concentration 10 mM), the mixture was left to stand at 0 ° C. for exactly 1 minute. By adding Mg²⁺, the cell membranes aggregate and precipitate with the exception of the brush border membranes. After centrifugation at 3000 xg (5000 rpm, SS-34 rotor) for 15 minutes, the precipitate was discarded and the supernatant, which contained the brush border membranes, was centrifuged at 267000 xg (15000 rpm, SS-34 rotor) for 30 minutes. The supernatant was discarded, the precipitate was rehomogenized in 60 ml of 12 mM Tris / HCl buffer (pH 7.1) / 60 mM mannitol, 5 mM EGTA using a Potter Elvejhem homogenizer (Braun, Melsungen, 900 rpm, 10 strokes). After adding 0.1 ml of 1 M MgCl₂ solution and incubating for 15 minutes at 0 ° C., the mixture was centrifuged again at 3000 × g for 15 minutes. The supernatant was then centrifuged for another 30 minutes at 46,000 xg (15,000 rpm, SS-34 rotor). The precipitate was taken up in 30 ml of 10 mM Tris / Hepes buffer (pH 7.4) / 300 mM mannitol and resuspended homogeneously by 20 strokes in a Potter Elvejhem homogenizer at 1000 rpm. After centrifugation at 48,000 xg (20,000 rpm, SS-34 rotor) for 30 minutes, the precipitate was taken up in 0.5 to 2 ml of Tris / Hepes buffer (pH 7.4) / 280 mM mannitol (final concentration 20 mg / ml) and resuspended using a tuberculin syringe with a 27 gauge needle. The vesicles were either used immediately after preparation for transport studies or stored in 4 mg portions in liquid nitrogen at -196 ° C.

2nd Inhibition of Na⁺-dependent [³H] taurocholate uptake in brush border membrane vesicles of the ileum

The uptake of substrates in the brush border membrane vesicles described above was determined by means of the so-called membrane filtration technique. 10 µl of the vesicle suspension (100 µg protein) were pipetted as drops onto the wall of a polystyrene incubation tube (11 x 70 mm), which contained the incubation medium with the corresponding ligands (90 µl). The incubation medium contained 0.75 µl = 0.75 µCi [3 H (G)] taurocholate (specific activity: 2.1 Ci / mmol) / 0.5 µl 10 mM taurocholate / 8.75 µl sodium transport buffer ( 10 mM Tris / Hepes (pH 7.4) / 100 mM mannitol / 100 mM NaCl) (Na-TP) or 8.75 µl potassium transport buffer (10 mM Tris / Hepes (pH 7.4) / 100 mM mannitol / 100 mM KCl) (KTP) and 80 µl of the inhibitor solution in question, depending on the experiment, dissolved in Na-T buffer or KT buffer. The incubation medium was filtered through a polyvinylidene fluoride membrane filter (SYHV LO 4NS, 0.45 μm, 4 mm ⌀, Millipore, Eschborn, FRG). The transport measurement was started by mixing the vesicles with the incubation medium. The concentration of taurocholate in the incubation mixture was 50 µM. After the desired incubation time (usually 1 minute), the transport was stopped by adding 1 ml of ice-cold stop solution (10 mM Tris / Hepes (pH 7.4) / 150 mM KCl). The resulting mixture was immediately sucked off under a vacuum of 25 to 35 mbar through a membrane filter made of cellulose nitrate (ME 25, 0.45 µm, 25 mm diameter, Schleicher &amp; Schuell, Dassell, FRG). The filter was washed with 5 ml of ice-cold stop solution.

To measure the uptake of the radioactively labeled taurocholate, the membrane filter was dissolved with 4 ml of the Quickszint 361 scintillator (Zinsser Analytik GmbH, Frankfurt, FRG) and the radioactivity was measured by liquid scintillation measurement in a TriCarb 2500 measuring device (Canberra Packard GmbH, Frankfurt, FRG) . The measured values ​​were determined after calibration of the device using standard samples and after correction, if necessary Existing chemiluminescence obtained as dpm (decompositions per minute).

The control values ​​were determined in Na-TP and KTP, respectively. The difference between the uptake in Na-TP and KTP resulted in the Na-dependent transport share. The IC₅₀ Na⁺ was the concentration of inhibitor at which the Na⁺-dependent transport fraction was inhibited by 50% - based on the control.

The pharmacological data comprise a series of tests in which the interaction of the compounds according to the invention with the intestinal bile acid transport system in the terminal small intestine was investigated. The results are summarized in Table 1.

The invention further relates to the use of the compounds according to the invention for the production of a medicinal product. For this purpose, the compounds of formula I are dissolved or suspended in pharmacologically acceptable organic solvents, such as mono- or polyhydric alcohols, such as. As ethanol or glycerol, in triacetin, oils, e.g. B. sunflower oil, cod liver oil, ethers, such as. B. diethylene glycol dimethyl ether, or polyethers, for. B. Polyethylene glycol, or in the presence of other pharmacologically acceptable polymer carriers, such as. B. polyvinyl pyrrolidone, or other pharmaceutically acceptable additives such as starch, cyclodextrin or polysaccharides. Furthermore, the compounds according to the invention can be given in combination with other pharmaceutical substances.

The compounds of formula I are administered in various dosage forms, preferably orally in the form of tablets, capsules or liquids. The daily dose, depending on the body weight and constitution of the patient, is in the range from 3 mg to 5000 mg, but preferably in the dose range 10 to 1000 mg.

example 1

<chemistry id="chem0017" num="0017"><img file="EP0624596A2_D0017.tif" /></chemistry> 5.0 g (10.9 mmol) of triformyl compound (J. Lip. Res. 29, 1387, 1988) are deprotected in 100 ml of 1M NaOMe / MeOH solution for 2 hours at room temperature. For working up, water is added and the methanol is stripped off. It is extracted three times with CH₂Cl₂, dried over MgSO₄ and concentrated. 3.9 g (96%) of the unprotected nitrile are obtained. MS (FAB, 3-NBA / LiCl) C₃₃H₃₇NO₃ (375) 383 (M + Li⁺)

Example 2

<chemistry id="chem0018" num="0018"><img file="EP0624596A2_D0018.tif" /></chemistry> 3.8 g (10.1 mmol) of trihydroxy compound (Example 1) are dissolved in 40 ml of CH₂Cl₂, 20 ml of dihydropyran and 300 mg of pyridinium (toluene-4-sulfonate) are added at 0 ° C. and the mixture is then stirred for 2 days at room temperature . The reaction mixture is concentrated and the residue is chromatographed on silica gel (cyclohexane / ethyl acetate 2: 1). 5.7 g (90%) of product are obtained. MS (FAB, 3-NBA / LiCl) C₃₃H₅₃NO₅ 8543) 550 (M + Li⁺)

Example 3

<chemistry id="chem0019" num="0019"><img file="EP0624596A2_D0019.tif" /></chemistry> 2.0 g (3.19 mmol) of nitrile (Example 2) and 2.1 g (6.34 mmol) of tributyltin azide are refluxed in toluene for 6 days. After 3 days, a further 2.1 g of tributyltin azide are added. The reaction mixture is concentrated. After chromatography of the residue (silica gel, ethyl acetate / MeOH 95: 5), 1.7 g (79%) of tetrazole derivative are obtained. MS (FAB), 3-NBA / LiCl) C₃₈H₆₂N₄O₆ (670) 677 (M + Li⁺)

Example 4

<chemistry id="chem0020" num="0020"><img file="EP0624596A2_D0020.tif" /></chemistry> 1.6 g (2.39 mmol) of THP-protected compound (Example 3), 200 mg of pyridinium p-toluenesulfonate and 1.6 ml of acetic acid are refluxed in 30 ml of methanol for 12 hours. After cooling, the mixture is concentrated and the residue is chromatographed on silica gel (CH₂Cl₂ / MeOH 9: 1). Yield: 720 mg (87%) MS (FAB, 3-NBA / LiCl) C₂₃H₃₈N₄O₃ (418) 425 (M + Li⁺)

Example 5

<chemistry id="chem0021" num="0021"><img file="EP0624596A2_D0021.tif" /></chemistry> 21 g (36.6 mmol) iodine compound (tetrahedron, <u>45</u> (17), 5423, 1989) and 3.6 g of sodium cyanide are stirred in 300 ml of DMSO at 50 ° C. for 1.5 hours. The reaction mixture is poured onto ice water and extracted with ethyl acetate. The organic phase is dried (MgSO₄) and concentrated. Chromatography (cyclohexane / ethyl acetate) provides 15 g (87%) of nitrile. MS (FAB, 3-NBA / LiCl) C₂₇H₃₉NO₆ (473) 480 (M + Li⁺)

Example 6

<chemistry id="chem0022" num="0022"><img file="EP0624596A2_D0022.tif" /></chemistry> The formyl-protected compound (Example 5) is deprotected according to the procedure described for Example 1. MS (FAB, 3-NBA / LiCl) C₂₄H₃₉NO₃ (389) 396 (M + Li⁺) Examples 7, 8 and 9 were prepared by the procedure described for Examples 2, 3 and 4.

Example 7

<chemistry id="chem0023" num="0023"><img file="EP0624596A2_D0023.tif" /></chemistry> MS (FAB, 3-NBA / LiCl) C₃₉H₆₃NO₆ (641) 648 (M + Li⁺)

Example 8

<chemistry id="chem0024" num="0024"><img file="EP0624596A2_D0024.tif" /></chemistry> MS (FAB, 3-NBA / LiCl) C₃₉H₆₄N₄O₆ (684.5) 691.6 (M + Li⁺)

Example 9

<chemistry id="chem0025" num="0025"><img file="EP0624596A2_D0025.tif" /></chemistry> MS (FAB, 3-NBA / LiCl) C₂₄H₄₀N₄O₃ (432) 439 (M + Li⁺) The compound of Example 9 can also be obtained directly from Example 6 and tributyltin azide (see Example 3). The crude product is chromatographed twice for purification (1st ethyl acetate / MeOH 9: 1; 2nd CH₂Cl₂ / MeOH / acetic acid 9: 1: 0.1). The yield is 83%.

> Example 10

<chemistry id="chem0026" num="0026"><img file="EP0624596A2_D0026.tif" /></chemistry> 100 g (215 mmol) of trihydroxy compound (EP-A-0 489 423) are protected according to Example 2 with tetrahydropyranyl groups. The crude product obtained is reacted without further purification. Dissolved in 250 ml ether, it is slowly added dropwise at 0 ° C. to a suspension of 15 g LiAlH₄ in 500 ml ether. After 2 hours at 0 to 5 ° C, water is carefully added and then extracted several times with ether. The organic phase is dried (MgSO₄) and concentrated. 100 g (67%) of product are obtained.

Example 11

<chemistry id="chem0027" num="0027"><img file="EP0624596A2_D0027.tif" /></chemistry> 20 g (29.0 mmol) of the alcohol (Example 10) are dissolved in 150 ml of pyridine. 2.45 ml (31.0 mmol) of methanesulfonic acid chloride are slowly added dropwise at 0 ° C. and the mixture is then stirred at room temperature for 2 hours. For working up, it is poured onto ice water and extracted with ethyl acetate. The organic phase is dried (MgSO₄) and concentrated. The crude product is dissolved in 200 ml of DMSO, 3.0 g of NaCN are added and the mixture is stirred at 50 ° C. for 1 hour. It is poured onto ice water and extracted with ethyl acetate. After the organic phase has dried, the mixture is concentrated and chromatographed (cyclohexane / ethyl acetate 7: 3). 15 g (77%) of nitrile are obtained. MS (FAB, 3-NBA / LiCl) C₄₂H₆₉NO₇ (699) 706 (M + Li⁺)

Example 12

<chemistry id="chem0028" num="0028"><img file="EP0624596A2_D0028.tif" /></chemistry> Example 12 was obtained by the procedure described for Example 11. Examples 13 to 16 are prepared according to the procedures described for Examples 3 and 4.<chemistry id="chem0029" num="0029"><img file="EP0624596A2_D0029.tif" /></chemistry><tables id="tabl0001" num="0001"><table frame="all"><tgroup cols="4" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="39.37mm" /><colspec colnum="2" colname="col2" colwidth="39.37mm" /><colspec colnum="3" colname="col3" colwidth="39.37mm" /><colspec colnum="4" colname="col4" colwidth="39.37mm" /><thead valign="top"><row><entry namest="col1" nameend="col1" align="center">example</entry><entry namest="col2" nameend="col2" align="center">R</entry><entry namest="col3" nameend="col3" align="center">n</entry><entry namest="col4" nameend="col4" align="center">MS (FAB, 3-NBA / LiCl)</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="right">13</entry><entry namest="col2" nameend="col2" align="center">THP</entry><entry namest="col3" nameend="col3" align="right">0</entry><entry namest="col4" nameend="col4" align="center">C₄₀H₆₆N₄O₆ (698.5) 705.7 (M + Li⁺)</entry></row><row><entry namest="col1" nameend="col1" align="right">14</entry><entry namest="col2" nameend="col2" align="center">H</entry><entry namest="col3" nameend="col3" align="right">0</entry><entry namest="col4" nameend="col4" align="center">C₂₅H₄₂N₄O₃ (446) 453 (M + Li⁺)</entry></row><row><entry namest="col1" nameend="col1" align="right">15</entry><entry namest="col2" nameend="col2" align="center">THP</entry><entry namest="col3" nameend="col3" align="right">1</entry><entry namest="col4" nameend="col4" align="center">C₄₂H₇₀N₄O₇ (742.5) 749.4 (M + Li⁺)</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="right">16</entry><entry namest="col2" nameend="col2" align="center">H</entry><entry namest="col3" nameend="col3" align="right">1</entry><entry namest="col4" nameend="col4" align="center">C₂₇H₄₆N₄O₆ (490) 497 (M + Li⁺)</entry></row></tbody></tgroup></table></tables>

Example 17

<chemistry id="chem0030" num="0030"><img file="EP0624596A2_D0030.tif" /></chemistry> 15 g (30.6 mmol) of the alcohol from Example 16 are dissolved in 200 ml of CH₂Cl₂, mixed with 100 ml of pyridine and reacted at -20 to -10 ° C for 2.5 hours with 5 ml of methanesulfonic acid chloride. The still cold reaction solution is poured onto ice water, extracted with ethyl acetate, the organic phase dried (MgSO₄) and concentrated. The crude product is purified on a short silica gel column (CHCl₃ / MeOH 93: 7). 16 g (92%) of product are obtained. MS (FAB, 3-NBA / LiCl) C₂₈H₄₈N₄O₆S (568) 575 (M + Li⁺)

Example 18

<chemistry id="chem0031" num="0031"><img file="EP0624596A2_D0031.tif" /></chemistry> 2.3 g (84.05 mmol) of mesyl compound (Example 17) and 290 mg of sodium azide are stirred in 30 ml of DMF at 100 ° C. for 2 hours. After cooling, the reaction mixture is concentrated and the residue is chromatographed on silica gel (CH₂Cl₂ / MeOH 85/15). The yield is 1.0 g (49%). MS (FAB, 3-NBA / LiCl) C₂₇H₄₅N₇O₃ (515) 522 (M + Li⁺)

Example 19

<chemistry id="chem0032" num="0032"><img file="EP0624596A2_D0032.tif" /></chemistry> 1.0 g (1.94 mmol) of azido compound are dissolved in 30 ml of methanol / 1.5 ml of water and hydrogenated in the presence of 50 mg of Pd black with H₂. It is filtered off from the catalyst and concentrated. The residue is chromatographed on silica gel (CH₂Cl₂ / MeOH / NEt₃ 8: 2: 1) and 550 mg (58%) amine are obtained. MS (FAB, 3-NBA / LiCl) C₂₇H₄₇N₅O₃ (489) 496 (M + Li⁺)

Example 20

<chemistry id="chem0033" num="0033"><img file="EP0624596A2_D0033.tif" /></chemistry> 3.0 g (3.76 mmol) of the unprotected compound (EP-A-0 489 423) are dissolved in 30 ml of formic acid, 0.2 ml of perchloric acid are added and the mixture is stirred at 50 ° C. for 2 hours. 30 ml of acetic anhydride are then added dropwise at room temperature, and the mixture is stirred for a further 30 minutes. It is poured onto ice water and extracted with ethyl acetate. The organic phase is dried, concentrated and the residue is chromatographed. 1.93 g (55%) of formyl-protected compound are obtained. MS (FAB, 3-NBA) C₅₃H₇₉NO₁₃ (938) 939 (M + H⁺)

Example 21

<chemistry id="chem0034" num="0034"><img file="EP0624596A2_D0034.tif" /></chemistry> 0.5 g (0.53 mmol) of Example 20 are dissolved in 30 ml of dry THF, 130 mg (0.62 mmol) of PCl₅ are added and the mixture is stirred at room temperature for 30 minutes. A solution of 200 mg (2.35 mmol) of anhydrous 5-aminotetrazole in 10 ml of DMF is added to the reaction solution. After a further 3 hours at room temperature, the mixture is concentrated and the residue is chromatographed (CHCl₃ / MeOH 9: 1). 400 mg (75%) of tetrazole compound are obtained. MS (FAB, 3-NBA / LiCl) C₅₄H₈₀N₆O₁₂ (1005) 1012 (M + Li⁺)

Example 22

<chemistry id="chem0035" num="0035"><img file="EP0624596A2_D0035.tif" /></chemistry> To split off the formyl groups, 370 mg (0.37 mmol) of Example 21 are dissolved in 20 ml of EtOH, 2 ml of 1N NaOH solution are added and the mixture is stirred at room temperature for 6 hours. It is then concentrated, water is added and 1N HCl to bis pH 2 is added. The precipitate is filtered off, dissolved in methanol, filtered and concentrated again. 170 mg (53%) of product are obtained. MS (FAB, 3-NBA / LiCl] C₄₉H₈₀N₆O₇ (865) 872 (M + Li⁺).

Examples 23 and 24 are prepared from triformylcholic acid by the methods described for Examples 21 and 22.<chemistry id="chem0036" num="0036"><img file="EP0624596A2_D0036.tif" /></chemistry><tables id="tabl0002" num="0002"><table frame="all"><tgroup cols="3" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="52.50mm" /><colspec colnum="2" colname="col2" colwidth="52.50mm" /><colspec colnum="3" colname="col3" colwidth="52.50mm" /><thead valign="top"><row><entry namest="col1" nameend="col1" align="center">example</entry><entry namest="col2" nameend="col2" align="center">R</entry><entry namest="col3" nameend="col3" align="center">MS (FAB, 3-NBA / LiCl)</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="right">23</entry><entry namest="col2" nameend="col2" align="center">-CHO</entry><entry namest="col3" nameend="col3" align="left">C₂₈H₄₁N₅O₇ (559) 566 (M + Li⁺)</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="right">24</entry><entry namest="col2" nameend="col2" align="center">H</entry><entry namest="col3" nameend="col3" align="left">C₂₅H₄₁N₅O₄ (475) 482 (M + Li⁺)</entry></row></tbody></tgroup></table></tables>

Example 25

<chemistry id="chem0037" num="0037"><img file="EP0624596A2_D0037.tif" /></chemistry> 150 mg (0.31 mmol) of amino compound (Example 19), 130 mg (0.32 mmol) of cholic acid, 80 mg (0.64 mmol) of hydroxybenzotriazole and 65 mg (0.32 mmol) of dicyclohexylcarbodiimide are mixed in 20 ml of THF for 24 hours stirred at room temperature. The reaction mixture is concentrated and the residue is chromatographed on silica gel (CH₂Cl₂ / MeOH / NEt₃ 8: 2: 1). 250 mg (88%) of product are obtained. MS (FAB, 3-NBA / LiCl) C₅₁H₈₅N₅O₇ (880) 887 (M + Li⁺) Examples 26 and 27 are obtained by the procedure described for Example 25.<chemistry id="chem0038" num="0038"><img file="EP0624596A2_D0038.tif" /></chemistry><tables id="tabl0003" num="0003"><table frame="all"><tgroup cols="3" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="52.50mm" /><colspec colnum="2" colname="col2" colwidth="52.50mm" /><colspec colnum="3" colname="col3" colwidth="52.50mm" /><thead valign="top"><row><entry namest="col1" nameend="col1" align="center">example</entry><entry namest="col2" nameend="col2" align="center">R</entry><entry namest="col3" nameend="col3" align="center">MS (FAB, 3-NBA / LiCl)</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="right">26</entry><entry namest="col2" nameend="col2" align="left">α-OH</entry><entry namest="col3" nameend="col3" align="left">C₅₁H₈₅N₅O₆ (864) 865 (M + H⁺)</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="right">27</entry><entry namest="col2" nameend="col2" align="left">β-OH</entry><entry namest="col3" nameend="col3" align="left">C₅₁H₈₅N₅O₆ (864) 865 (M + H⁺)</entry></row></tbody></tgroup></table></tables>

Example 28

<chemistry id="chem0039" num="0039"><img file="EP0624596A2_D0039.tif" /></chemistry> 2.0 g (4.9 mmol) of cholic acid and 5 ml (36 mmol) of triethylamine are dissolved in 150 ml of THF, and 1.5 ml (16 mmol) of ethyl chloroformate are added at 0 ° C. After 15 minutes, 1.5 g (16 mmol) of aminoacetonitrile hydrochloride are added. It is stirred for 5 hours at room temperature. The resulting precipitate is filtered off and the solution is concentrated. After chromatography of the crude product (chloroform / methanol 17: 3), 1.8 g of product (85%) are obtained. MS (FAB, 3-NBA / LiCl) C₂₆H₄₂N₂O₄ (446) 453 (M + Li⁺)

Example 29

<chemistry id="chem0040" num="0040"><img file="EP0624596A2_D0040.tif" /></chemistry> 1.5 g (3.36 mmol) of the nitrile and 3.5 g (10.5 mmol) of tributyltin azide are heated under reflux in 100 ml of toluene for 24 hours. After the reaction is concentrated in vacuo and the residue chromatographed on silica gel (CHCl₃ / MeOH 7: 3). 830 mg (56%) of product are obtained. MS (FAB, 3-NBA) C₂₆H₄₃N₅O₄ (489) 490 (M + H⁺) Examples 30 and 31 were prepared using the procedures described for Examples 28 and 29.<chemistry id="chem0041" num="0041"><img file="EP0624596A2_D0041.tif" /></chemistry><tables id="tabl0004" num="0004"><img file="EP0624596A2_D0042.tif" /></tables>

Table 1 shows measured values ​​of the inhibition of the [3 H] taurocholate uptake in brush border membrane vesicles of the rabbit ileum. The quotients from the IC₅₀ or IC are given_{50 Well}-Values ​​of the reference substance taurochenodeoxycholate (TCDC) and the respective test substance.<tables id="tabl0005" num="0005"><img file="EP0624596A2_D0043.tif" /></tables>