DE2808006C2 - - Google Patents

Description

When studying the biological role of prostaglandins only recently has the mechanism been clarified, which in the organism of the Mammals regulates the process of blood clotting (chemical and engineering News, 20. XII. 1976, p. 17).

Thereafter, the common precursor of prostaglandins in their biosynthesis in the mammalian organism, prostaglandin endoperoxide, partly through an enzyme produced by the platelets (thrombocytes) to a very active compound that causes blood clotting, thromboxane A₂, and otherwise with the help of an in the enzyme that occurs in the aorta wall to form a very active compound that prevents blood clotting. The latter is - as the chemical structure determination showed - the 5- (Z) -9-deoxy-6.9 α- oxydo- Δ ⁵-PGF₁ _α , which was given the name prostacyclin (PGI₂).

The prostacyclin was biochemically derived from the prostaglandin endoperoxide with the help of mammalian aortic tissue obtained enzyme system. Its biological effectiveness was also detected in vitro, whereby it was shown that the by the adenosine diphosphate-induced platelet aggregation of the connection was interrupted or prevented (prostaglandines, Vol. 12, No. 5, 685 (1976)).

The invention relates to a method for producing Prostacyclin (PGI₂) and its derivatives of the general formula

wherein

R¹hydrogen, C₁- to C₁₀-alkyl, which by C₆- to C₁₀-aryl, amino, Hydroxyl or halogen may be substituted, or the equivalent a physiologically compatible organic or inorganic Cations, R²hydrogen, hydroxyl or protected hydroxyl, R³hydrogen or C₁- to C₆-alkyl, R⁴hydrogen or a hydroxyl protecting group, Straight or branched C₁ to C₁₀ alkyl, which by -O- or -NH- interrupted and / or substituted by a C₆ to C₁₀ aryl can,

mean, characterized in that in each case in a known per se wise

• a) PGF₂ _α or its derivatives of the general formula whereinR², R³, R⁴ and R⁵ have the meanings given, and
  R⁶hydrogen, C₁- to C₁ Alkyl-alkyl, which can be substituted by C₆- to C₁₀-aryl, amino, hydroxyl or halogen, the equivalent of a physiologically compatible organic or inorganic cation or a hydroxyl protecting group, in a solvent with a brominating agent for reaction brings,
• b) from the 5-bromo-PGI 1 thus obtained or its derivatives of the general formula wherein R², R³, R⁴, R⁵ and R⁶ have the meanings given, splits off hydrogen bromide and
• c) in the PGI₂ thus obtained or its derivatives of general Formula I, if desired, the hydroxyl protecting groups in the Substituents R⁶ and / or R⁴ and / or R² removed, the substituents R¹ in a physiologically acceptable organic or inorganic cation or by esterification in a C₁ to C₁₀ alkyl, which by C₆ to C₁₀ aryl, amino, Hydroxyl or halogen may be substituted, or converted to hydrogen by hydrolysis of the ester

The prostacyclin mentioned at the beginning - also in the specialist literature as Designated PGI₂ - is a compound of general formula I, in the R¹, R³ and R⁴ are hydrogen, R² is hydroxyl and R⁵ is n-pentyl. If R¹ is methyl, the PGI₂ methyl ester is present; R¹ is one physiologically compatible cation, so it is about PGI₂ salts.

Under the alkyl group R¹ or R⁶ is one optionally by C₆- bis C₁₀ aryl, amino, hydroxyl or halogen substituted C₁ to C₁₀ alkyl group to understand. The preference will be a straight or branched alkyl with 1-6 carbon atoms, especially with 1-4 carbon atoms, e.g. B. the methyl, ethyl, n and Isopropyl, n, iso, sec. and tert-butyl group. At the as Substituent of this alkyl group possible C₆ to C₁₀ aryl group it is a unsubstituted aryl group, such as the phenyl and 1- or 2-naphthyl group. The alkyl group R¹ or R⁶ can be replaced by one or several of the groups mentioned anywhere be substituted. For water solubility, it is advantageous when R¹ or R⁶ the equivalent of a physiologically acceptable means inorganic or organic cations. Some examples for organic cations, the different primary ones are secondary, tertiary and quaternary ammonium cations, and for inorganic cations the alkali and alkaline earth cations, sodium, Lithium, potassium, ammonium, trishydroxymethyl methylammonium,  Piperidinium, pyrrolidinium and morpholinium ions are preferred.

If R² is a protected hydroxyl group, the protective group can either be different from R⁴ or correspond to R⁴. The latter is preferred. Protecting groups for the hydroxyl groups are all protective groups customary for this purpose in prostaglandin chemistry. Some examples are:
the tetrahydropyranyl group,
the various alkylsilyl, the alkanoyl and the aroyl groups and
in particular the lower alkanoyl groups, such as the acetyl group,
furthermore the benzoyl and p-phenylbenzoyl group,
the various carbamoyl groups, such as the methylcarbamoyl, ethylcarbamoyl, phenylcarbamoyl and p-phenylphenylcarbamoyl groups.

The C₁ to C₆ alkyl group R³ can be straight or branched. they preferably has 1-4 carbon atoms. Some examples for this are: the methyl, ethyl, n and isopropyl, n, iso, sec.- and tert-butyl, the pentyl and the hexyl group.

R⁵ means a, optionally interrupted by -O- or -NH-, straight or branched alkyl group, the 1-10, preferably 1-6 Has carbon atoms. Some examples are: the various decyl, nonyl, octyl, heptyl, hexyl, pentyl, Butyl and propyl groups, also the methyl and the ethyl group. The alkyl group can be a C₆ to C₁₀ aryl group contain. Examples include: the Phenyl, 1- and 2-naphthyl group.

The hydroxyl protecting group R⁶ can have the same meaning that has been specified for the protective group of R² and for R⁴.

The compounds of general formula II are easily accessible compounds. The prostaglandin F _{2 α} (PGF _{2 α} ), ie the compound in which R⁶, R³ and R⁴ are hydrogen, R² hydroxyl R⁵ n-pentyl, and their salts are commercially available as pharmaceuticals. Numerous processes are also known for producing the PGF _{2 α} esters. According to process step a), the compounds of the general formula II are treated in a solvent with a brominating agent. All brominating agents known from the literature can be used as brominating agents, e.g. B. elemental bromine, complex-bound bromine, preferably the pyridine bromine complex, and the various known N-bromine reagents, such as N, N-dibromodimethylhydantoin, N-bromoacetamide, N-bromosuccinimide, or the tribromide ions (Br ^- ₃) supplying reagents.

The bromination can be carried out in both an acidic and an alkaline medium be carried out. Any inert solvent can be used organic, protic or aprotic solvents are used will. Alkanols and organic ones come as protic solvents Acids, as aprotic solvents, acetonitrile, dimethylformamide, Dimethyl sulfoxide, chlorinated hydrocarbons and ether in question. Water can also be used as a solvent. The solvents can be used individually or as mixtures. The Bromination is, depending on the reactivity of the reagents used, at temperatures between -78 and + 100 ° C, preferably carried out at room temperature. The course of the reaction can be followed well by thin layer chromatography.

The specified brominating agents attack the Δ ⁵-cis double bond of the compounds of the general formula II regioselectively, the corresponding bromide ion being formed. The resulting, positively charged ions stabilize in any case with the inclusion of the hydroxyl group in the 9-position, 5-bromo-PGI₁ or its derivatives of the general formula III

form in which R² to R⁶ have the meanings given.  

The in the implementation of the compounds of the invention general formula II with brominating compounds the general formula III are in two isomeric forms, the with regard to the spatial position of the hydrogen on Carbon atom 6 (according to the usual for prostaglandic acid Numbering) are to be addressed as epimers (exo-endo-isometry). By using different brominating agents and depending on the type of reagents and the reaction conditions Endo products are obtained in which the exo and the endo form of the compounds of general formula III in ratios of 1: 1 to 1:10. If desired, the epimer mixture can e.g. B. column chromatography on silica gel. These Separation is particularly easy with the esters of the compounds perform the general formula III, in which R⁶ a optionally by aryl, amino, hydroxyl or halogen substituted alkyl group or a suitable hydroxyl protecting group means.

If the PGF _{2 α-} acid or its salts are subjected to bromination as the starting compounds of the general formula II, the compounds of the general formula III obtained can be esterified in a manner known per se. The methyl esters of the general formula III (in which R⁶ is methyl) are most easily obtained by reacting with diazomethane in ether.

The elimination of hydrogen bromide from the 5-bromo-PGI₁ derivatives of General formula III according to process stage b) is advantageously in carried out in such a way that the compounds of general Formula III optionally treated with a base with heating. A preferred embodiment of this reaction is that to use the compound of general formula II in an alkanol Reacts alkali alcoholates or together with tertiary aliphatic or aromatic bases, e.g. B. pyridine, picoline or triethylamine warmed up. Others can respond to the reaction  Elimination reactions well-known reagents, e.g. B. Diazabicyclo-undecane (DBU) or diazabicyclo-nonane (DBN) in the usual solvents are used.

In order to avoid side reactions, a is preferably used as the base Reagent that is weaker than the specified bases, e.g. B. Potassium carbonate in an alkanol as a solvent used. The Hydrogen bromide is expedient at temperatures between Room temperature and 200 ° C carried out.

The elimination reaction in the endo isomer of the 5-bromo-PGI₁ derivative the general formula III is much faster as the exo isomer, as the end product of the elimination however, with both isomers the 5 (Z) isomer of the PGI₂ derivatives general formula I obtained.

Process stage c), from the PGI₂ derivatives obtained in process stage b) of general formula I - if desired - the Hydroxyl protecting groups of the substituents R⁶, R⁴ and / or R² remove the substituent R¹ in a physiologically acceptable Cation to convert to an ester or hydrogen is in performed in a known manner.

The PGI₂ derivatives of the general formula I are in the usual way isolated from the reaction mixture. Since they are one against acid contain extremely sensitive enol ether structure it suitably at low temperatures with the addition of a small amount of base, preferably sodium carbonate, stored.

The PGI₂ derivatives of general formula I obtained according to the invention have the property in a concentration of 0.2 to 20 ng / ml the z. B. aggregation triggered by adenosine diphosphate to prevent human platelets or one already dissolve thrombus formed.

The aggregation-inhibiting or thrombus-dissolving action of the PGI₂ derivatives of the general formula I is shown using the example of the PGI₂ methyl ester prepared according to the invention from the PGF _{2 α} -methyl ester, ie a compound of the general formula I in which R³ and R⁴ are hydrogen, R² hydroxyl , R¹ is methyl and R⁵ is n-pentyl.

For the investigation of aggregation-inhibiting or thrombus-releasing Platelet rich plasma (PRP) effect was used by each 1 ml examined in a modified Born aggregometer has been.

• 1) The aggregation of the control sample was triggered 1 × 10 ^-5 mol adenosine diphosphate. The curve shows the measured aggregation (in divisions of scale) as a function of time.-.-.- in FIG. 1.
• 2) 5 to 20 ng of PGI₂ methyl ester were dissolved in 0.05 molar TRIS hydrochloric acid buffer, pH 7.5, and incubated together with 1 ml of PRP at room temperature for 1.5 minutes. The solution was then introduced into the aggregometer and the aggregation was induced by adding 1 × 10 ^-5 mol of adenosine diphosphate. An inhibition was observed during the aggregation. The aggregation as a function of time is illustrated by curve xxxx-.
• 3) The aggregation triggered by adenosine diphosphate could be reversed by 50% by adding 20 ng of PGI₂ methyl ester. The result of this investigation is illustrated in Fig. 1 by curve xxx.

The process of the invention is illustrated by the following examples explained.

example 1

290 mg (0.82 mmol) of PGF _{2 α are dissolved} in 2 ml of acetonitrile and 140 mg (0.52 mmol) of N, N′-dibromodimethylhydantoin are added to the resulting solution with stirring at 0 ° C. The reaction is complete within 5 minutes, the compounds: 6-endo-6.9 α- oxydo-5 α -bromo-11 α , 15 α -dihydroxy-13 (E) -prostenic acid and 6-exo-6 , 9 α- oxydo-5 β -bromo-11 α , 15 α -dihydroxy-13 (E) -prostenic acid form approximately in a ratio of 5: 1. They contain minimal or greater polarity impurities in minimal amounts. On silica gel, with a solvent mixture of benzene, dioxane and acetic acid in a ratio of 20: 10: 1, the endo compound has an R _f value of 0.22 and the exo compound has an R _f value of 0.27 .

The 430 mg obtained after removal of the solvent Crude product are column chromatographed on silica gel (Mixed solvent: chloroform: acetic acid = 20: 5: 1) in pure exo compound (31.1 mg), a mixture of exo and endo compound (94.5 mg) and pure endo compound (102 mg) separated.

The pure isomers or the mixture of isomers can be converted into the corresponding methyl esters by esterification with diazomethane in ether at 0 ° C. in approximately quantitative yield. In thin-layer chromatography under the conditions mentioned above, the methyl esters have the following R _f values:
Endo connection: 0.35,
Exo connection: 0.41.

Example 2

175 g (0.49 mmol) of PGF₂ _{α are dissolved} in 5 ml of dichloromethane and 141 mg (0.55 mmol) of N-bromocampherimide are added to the resulting solution at room temperature with stirring. The reaction is complete within 3 hours, the compounds: 6-endo-6.9 α -oxydo-5 α -bromo-11 a , 15 α -dihydroxy-13 (E) -prostenic acid and 6-exo-6 , 9 α- oxydo-5 β -bromo-11 α , 15 α -dihydroxy-13 (E) -prostenic acid in a ratio of about 2: 1. The 320 mg of crude product obtained after removal of the solvent are purified by column chromatography in the manner described in Example 1. The following products are received:
Exo compound: 45 mg,
Mixture of endo and exo compound: 62 mg,
Endo compound: 60 mg.
The R _f values of the compounds correspond to the values given in Example 1.

Example 3

973 mg (2.74 mmol) of PGF _{2 α} are dissolved in 10 ml of dichloromethane and a solution of 535 mg (3.01 mmol) of N-bromosuccinimide in 5 ml of dichloromethane is added with stirring. The reaction is complete within 80 to 90 minutes. The reaction produces endo and exo isomers in a ratio of about 4: 1. The compounds are separated by column chromatography into the following fractions in the manner described in Example 1:
131 mg exo compound,
578 mg mixture of exo and endo compound and
293 mg endo compound.
The R _f values of the compounds correspond to the values given in Example 1.

Example 4

251 mg of PGF _{2 α are dissolved} in a mixture of 10 ml of water and 10 ml of acetic acid, and first the solution of 1 g of potassium bromide in 5 ml of water and then the solution of 40 mg of potassium bromate in 5 ml of water are added to the solution. The reaction is complete within 3 to 4 hours.

The reaction mixture is extracted with 20 ml of ether, the ethereal phase is washed first with 0.5 ml of 10% sodium thiosulfate solution, then 4 times with 5 ml of water and finally dried at 0 ° C. with exclusion of light over magnesium sulfate. The ethereal solution is then filtered, evaporated and the residue is freed from acetic acid at 133.3 Pascal to constant weight. 342 mg of a compound corresponding mainly to formula III are obtained. This crude product is purified in the manner described in Example 1 and subjected to column chromatography. The following fractions are obtained:
6 mg exo compound,
103 mg mixture of exo and endo compound and
72 mg endo compound.
The R _f values of the products correspond to the values given in Example 1.

Example 5

278 mg (0.62 mmol) of 6.9 α -oxydo-5-bromo-11 α , 15 α -dihydroxy-13 (E) -prostenic acid methyl ester are dissolved in 5 ml of absolute ethanol and 3 ml of a solution are added to the solution given ethanolic potassium alcoholate concentration 0.4 mol / 1. The reaction mixture is stirred at 70 ° C. for 6 hours and then the ethanol is removed. 50 ml of ether and 20 ml of water are added to the residue at 0 ° C., the ethereal phase is separated off after shaking and dried at 0 ° C. over anhydrous potassium carbonate. After evaporation of the ether, 145 mg (64%) of 5 (Z) -9-deoxy-6.9 α- oxydo- Δ -PGF _{1 a} methyl ester (PGI₂ methyl ester) is obtained in the form of an oil.
NMR spectrum: 5.45 (2H, m), 4.55 (1H, m) ppm.
IR spectrum: γ _max = 1730 and 1765 cm ^-1 .

On a silica gel layer impregnated with a triethylamine / ether mixture, the product shows an R _f value of 0.58 with a mixture of acetone and ethyl acetate in a ratio of 1: 1 as the mobile phase.

Example 6

220 mg (0.51 mmol) 6.9 α -Oxydo-5-bromo-11 α , 15 α -dihydroxy-13 (E) - prostatic acid methyl ester are dissolved in 10 ml of absolute tert-butanol and added to the solution In an argon atmosphere, 340 mg (3.1 mmol) of potassium tert-butoxide were added. The solution is stirred at 60 ° C for 4 hours. The reaction mixture is then worked up further in the manner described in Example 5. 141 mg (75%) of PGI₂ methyl ester are obtained, the physical constants of which correspond to those of the product according to Example 5.

Example 7

233 mg (0.54 mmol) 6.9 α -Oxydo-5-bromo-11 α , 15 α -dihydroxy-13 (E) - prostate acid methyl ester are dissolved in 10 ml of absolute ethanol. 1 g of anhydrous potassium carbonate is added to the solution obtained, and the suspension is stirred at 70 ° C. for 12 hours. The reaction mixture is worked up further in the manner described in Example 5. 137 mg (72%) of PGI₂ methyl ester are obtained. The physical constants of the product correspond to those of the product according to Example 5.

Claims (4)

1. Process for the preparation of prostacyclin (PGI₂) and its derivatives of the general formula whereinR¹hydrogen, C₁- to C₁₀ alkyl, which can be substituted by C₆- to C₁₀ aryl, amino, hydroxyl or halogen, or the equivalent of a physiologically acceptable organic or inorganic cation, R²hydrogen, hydroxyl or protected hydroxyl, R³hydrogen or C₁- bis C₆-alkyl, R⁴hydrogen or a hydroxyl protecting group, R⁵ straight or branched C₁- to C₁₀-alkyl, which can be interrupted by -O- or -NH- and / or substituted by a C₆- to C₁₀-aryl, characterized in that one in a manner known per se

• a) PGF₂ _α or its derivatives of the general formula whereinR², R³, R⁴ and R⁵ have the meanings given, and
  R⁶hydrogen, C₁- to C₁ Alkyl-alkyl, which can be substituted by C₆- to C₁₀-aryl, amino, hydroxyl or halogen, the equivalent of a physiologically compatible organic or inorganic cation or a hydroxyl protecting group, in a solvent with a brominating agent for reaction brings,
• b) from the 5-bromo-PGI 1 thus obtained or its derivatives of the general formula whereinR², R³, R⁴, R⁵ and R⁶ have the meanings given, splits off hydrogen bromide and
• c) in the PGI₂ or its derivatives of the general formula I thus obtained, if desired, the hydroxyl protective groups in the substituents R⁶ and / or R⁴ and / or R² removed, the substituents R¹ in a physiologically compatible organic or inorganic cation or by esterification in a C₁- bis C₁₀-alkyl, which can be substituted by C₆- to C₁₀-aryl, amino, hydroxyl or halogen, or converted into hydrogen by hydrolysis of the ester.

2. The method according to claim 1, characterized in that element bromine, complex-bound elemental bromine, preferably the pyridine bromine complex, N-bromine reagents, preferably N, N-dibromodimethylhydantoin, N-bromoacetamide, N-bromosuccinimide, as bromination agent in stage a) or tribromide ions (Br ^- ₃) supplying reagents. 3. The method according to claim 1 or 2, characterized in that the elimination of hydrogen bromide in step b) by treatment with Alkaline alcoholates in an alkanol or with tertiary aliphatic or aromatic nitrogen bases, in particular pyridine, collidine, Picolin or triethylamine, optionally in the presence of a Solvent, and / or with heating, or with alkali bases, preferably alkali carbonates, especially potassium carbonate in one Solvent, preferably in an alkanol at temperatures between room temperature and 200 ° C.   4. The method according to any one of claims 1 to 3, characterized in that he for the preparation of the R¹ esters of PGI₂ or its derivatives general formula I. the 5-bromo-PGI₁ derivatives of the general formula III, in which R⁶ Is hydrogen in a manner known per se with an esterification reagent, preferably with a lower diazoalkane, esterified and from the thus obtained R1 ester cleaves off the hydrogen bromide.