NO783243L - LASALOCID DERIVATIVES. - Google Patents

Description

The present invention relates to lasalocid derivatives with the formula

where R means aryl, substituted aryl, heteroaryl, aralkyl, cycloalkyl, lower alkyl, lower alkenyl, caralkyloxy-lower alkyl, aminoalkyl, lower alkylaminoalkyl1, di-(lower alkyl)aminoalkyl, hydroxyalkyl1, alkoxyalkyl1, thioalkylalkyl, carboxy-lower -alkyl or hydrogen.

The invention further relates to a method for the production of the compounds I and pharmaceutical compositions based on the compounds I.

The term "alkyl" used here denotes, alone or in connection with other residues, a linear or branched hydrocarbon group with 1-16 C atoms. Preferred alkyl groups are lower alkyl groups. The term "lower" denotes groups with 1-7 C atoms. Examples of lower alkyl groups are methyl, ethyl, isopropyl and tertiary butyl.

The term "aryl" denotes an unsubstituted monocarbocyclic aromatic residue, such as phenyl.

in I

!_ I

I l! Examples of "substituted aryl" are alkyl-, carboxy-, halogen-,! alkoxy- or nitro-substituted phenyl, such as tolyl.

The term "halogen" or "halo" includes chlorine, bromine, fluorine and iodine.

A cycloalkyl residue preferably contains 4-7 C atoms, such as cyclohexyl.

The term "heteroaryl" denotes an unsubstituted mono-heterocyclic aromatic radical such as pyridyl or furyl.

The term "aralkyl" denotes a straight or branched alkyl group which is substituted by a mono-carbocyclic residue, such as benzyl.

The term "lower alkenyl" denotes a straight or branched hydrocarbon group having an olefinic double bond and 2-7 carbon atoms.

A preferred group of compounds of formula I are those in which R<1> is aryl, substituted aryl, aralkyl, cycloalkyl, lower alkyl, carboxy-lower alkyl or hydrogen. Particularly preferred are compounds of formula I in which R<1> is phenyl, chlorophenyl, cyclohexyl, benzyl or carboxymethyl. Most preferred are the compounds of formula I in which R<1> is phenyl, i.e. the compound 1-(phenylthiomethyl)-1-decarboxylasaloside.

The compounds of formula I are obtained according to the invention by reacting a compound of formula

2 3 where R and R are lower alkyl or together with nitrogen-

the atom a 5- to 6-membered heterocyclic ring which may contain a further heteroatom,

with a compound of formula R^SH, where R"^ has the above meaning, in the presence of a weak base.

In this reaction, a weak base is one that is sufficient to form the orthoquinone methide and the conjugate base of the present mercaptan, i.e. R 2 S without causing a retro-aldol cleavage of the lasalocid molecule. Suitable bases are alkali metal, e.g. sodium or potassium carbonates and bicarbonates, trialkylamines or pyridine. Suitable solvents for this reaction are alcohols, e.g. methanol or ethanol, high-boiling ethers, dioxane or tetrahydrofuran and ethyl acetate. The reaction temperature can lie between room temperature and reflux temperature, whereby the reflux temperature of the solvent used is preferred. Preferred reaction partners are thiophenol, p-chlorothiophenol, cyclohexyl mercaptan, benzyl mercaptan and mercaptoacetic acid, whereby a compound of formula I is obtained in which R"<*>" is phenyl, p-chlorophenyl, cyclohexyl, benzyl respectively. carboxymethyl. The starting material of formula II can be prepared from lasalocid, a compound of formula; ; by means of a Mannich reaction, more specifically by means of a mixture of a dialkylamine or a cyclic amine such as morpholine, piperidine or pyrrolidine and p-formaldehyde or aqueous formaldehyde. Suitable solvents for this reaction are C₁-C₂ alcohols, toluene, benzene, chlorinated hydrocarbons and ethers such as dioxane or tetrahydrofuran. The reaction temperature can vary from 60°C to the reflux temperature of the solvent used, whereby reflux temperature is preferred. The compound of formula III, lasalocid, also known as "antibiotic X-537 A", is a crystalline antibiotic produced by a streptomycin organism from a soil sample collected in Hyde Park, Massachusetts. Lyophilized samples of the culture bearing the laboratory designation-X-537 were deposited-in the United States Department of Agriculture, Agriculture Research Service, Northern Utilization Research and Development Division, Peoria, Illinois. The cultivar, given the identification number NRRL 3382 by the Agricultural Research Service, was made publicly available through NRRL. A replacement culture which is a closely related strain of the original deposited culture NRRL 3382, i.e. has both common lineage and morphological characteristics, was deposited with NRRL under the number NRRL 3382R.. This culture, like the original deposited culture NRRL 3382, is in the public domain available. ;Lasalocid (antibiotic X-537 A) is produced by growing the Streptomyces organism in an air-blown immersion culture whose pH is approximately neutral, i.e. approx. 6.5 to 7.5. The medium receives a nitrogen source such as yeast, a yeast derivative, maize flour, bean flour etc., whereby soybean flour is particularly preferred, and a carbohydrate source such as sugar, molasses etc., whereby raw sugar is particularly preferred. The fermentation is carried out at slightly higher temperatures, i.e. between 25°C and 35°C, an incubation temperature of approx. 28°C. After a 4- to 6-day incubation, the fermentation mash is filtered off and the antibiotic is obtained through extraction. The compound of formula I has antihypertensive activity. Using a standard as a control for comparison purposes, 1-(phenylthiomethyl)-1-decarboxylasalocid was examined for antihypertensive activity. The experiments were carried out with individual oral doses on DOCA-Na rats over a period of 5 days. After the 5-day period, the preparation was discontinued. Blood pressure measurements were carried out during the 5-day periodj I and then daily after cessation of the preparation until the blood pressure-*i et had adjusted to the starting value before the medication. The daily dosage was 10 mg/kg. The following results were obtained:

1-[(carboxymethyl)thiomethyl]-1-decarboxylasaloside, 1-(p-chloro-phenylthiomethyl)-1-decarboxylasaloside, 1-(benzylthiomethyl)-1-decarboxylasaloside and 1-(cyclohexylthiomethyl)-1-decarboxylasaloside was also investigated.

The results are reproduced in the following table. The results show that there are differences in the onset and duration of antihypertensive effects, but that the above-mentioned compounds have an antihypertensive activity when blood pressure is compared on the 1st day with the 5th day after discontinuation of the drug.

Example A I

Capsules may contain the following ingredients:

Approach:

1. The components 1, 3 and 5 are mixed.

2. Components-2 and 5 are dissolved in distilled water and/or alcohol, granulated to the correct consistency and ground.

3. Drying in oven.

4. Grinding and mixing with talc and magnesium stearate for 2 min.

5. Encapsulation.

Example 1 B

&

Tablets can have the following composition:

Procedure: I

1. Mixing the components 1 - 4 in a suitable device.

2. Granulation with distilled water to a suitable consistency.

3. Paint.

3. Drying in oven.

4. Grinding mixture with magnesium stearate for 3 minutes.

5. Pressing into tablets.

For use as hypertensive agents, the compound of formula I can be brought in the form of pharmaceutical preparations with inert pharmaceutical aids in a suitable dosage form for oral administration. Other dosage forms, e.g. the parenteral, is also possible. Examples of oral dosage forms are tablets, capsules, dragees, suspensions and solutions. As aids, inorganic or organic substances such as e.g. gelatin, albumin, lactose, starch, magnesium stearate, preservatives (stabilizers), lubricants, emulsifiers, salts for modification

of the osmotic pressure and buffers are used which are common in such preparations.

It has been shown that the subacute oral administration for the treatment of high pressure in warm-blooded beings e.g. DOCA-Na hypertensive rat, i.e. a dosage of up to 5 days followed by cessation of the preparation is the most. effectively, when the dosage range is between approx. 5 mg/kg and 100 mg/kg, especially between 5 mg/kg and 10 mg/kg. The chronic oral administration, i.e.

a dosage beyond 5 days is most effective when a low dosage level is used, i.e. lower than 0.1 mg/kg daily, e.g. 0.01 mg/kg to approx. 5 mg/kg per day. The dosage plans described above can also be used in the treatment of other cardiovascular diseases

symptoms such as Angina pectoris, Claudication and reduced blood flow in the brain. For the dosage, the dosage form for the chronic administration is particularly preferred, i.e. a dosage from less than 0.1 mg/kg per day up to approx. 5 mg/kg per day.

EXAMPLE 1

The Streptomyces strain NRRL 3382 R was grown in aerated deep culture in shake flasks. The substrate was adjusted to pH 6.5 - 7.5 by the addition of potassium hydroxide and then sterilized. In a fermentation tank, the substrate containing 2% soybean meal, 2% raw sugar, 0.5% corn-swell water and 0.1% K^IPO^ was inoculated with 5 - 10%

of an inoculum from a 3-day-old deep culture. The fermentation was carried out at 28° under positive air pressure during which per min-out and per 150 - 300 1 liquid an air flow of 140-280 1 maintained. The culture was harvested after 4 to 6 days of fermentation, filtered and the antibiotic separated by extraction as follows: 204 L of culture solution was filtered and the moist filter cake was slurried in 100 L of butyl acetate and stirred overnight at room temperature. The mixture was then filtered and the aqueous layer separated and discarded. The butyl acetate solution having 30 million Bazillus E units was concentrated under reduced pressure to 3 L, washed with 10% sodium carbonate solution and dried over sodium sulfate.

After further evaporation to 300 ml and dilution with 350 ml of petroleum ether (boiling point 50-60°), 41 g of solid containing 25 million Bazillus E units were isolated. This solid was then extracted in a Soxhlet apparatus for 40 hours with 4 l of petroleum ether (boiling point 50 - 60°). The extract was brought to dryness under reduced pressure, the crystalline residue slurried in petroleum ether and filtered to give 24.49 g of a mixture of 3-methyl-6-[7-ethyl-4-hydroxy-3,5-dimethyl- 6-oxo-7-Z5-ethyl-3-methyl-5-(5-ethyl-5-hydroxy-6-methyl-2-tetrahydropyranyl)-2-tetrahydrofuryl7~heptyl]salicylic acid (Lasalocid) and its salt. The mother liquor gave an additional 5.73 g of the antibiotic.

After recrystallization from ether-petroleum ether, the sodium-containing material was dissolved in ether and washed with dilute sulfuric acid to transfer it into the free acid. Removal of the ether gave an oily residue which crystallized from ethanol to give pure Lasalocid ethanolate. Several recrystallizations from ethanol did not change the melting point which remained indistinct at 100-109°C.

in I

Example 2I A mixture of 5 g of Lasalocid ethanolate, 2.5 g of paraformaldehyde and 2.5 g of diethylamine in 200 ml of toluene was heated for 90 minutes with stirring at reflux. During reflux heating, the water was collected in a trap. The solution obtained was diluted with ether, washed with water and with highly diluted (approx. 0.05 N) hydrochloric acid. The organic layer was dried, filtered and the solvent removed under reduced pressure, whereby a pale yellow (many times colorless resin) remained. This crude Mannich base, 1-(diethylaminomethyl)-1-decarboxylasaloside, contained toluene but was used directly in the next step.

Example 3

A solution of 9.7 g of the Mannich base prepared in example 2, 4.5 ml of thiophenol and 4 mg of triethylamine in 125 ml of ethanol was heated for 18 hours at reflux. The solvent was evaporated under reduced pressure and the residue taken up in methylene chloride. After washing with water and dilute sodium carbonate solution, the solution was dried and the solvent was evaporated. The crude product was purified by chromatography on 200 g of silica gel. With hexane/ethyl acetate (8:1) thiophenol and with hexane/ethyl acetate (6:1) the product, 1-(phenyl-thio-methyl)-1-decarboxylasaloside, were eluted. Evaporation of the solvent, finally under reduced pressure, gave a colorless foam.

Example 4

A solution of 1.4 g of 1-(diethylaminomethyl)-1-decarboxylasaloside,

2 g of p-chlorothiophenol and 2 ml of triethylamine in 50 ml of ethanol were heated for 18 hours at reflux. When the reaction was not complete (thin-layer chromatogram), a further 2 g of p-chlorothiophenol and 2 ml of triethylamine were added and reflux heating continued for 24 hours. After cooling, the solvent amine was evaporated under reduced pressure and the residue taken up in methylene chloride. This solution was washed with aqueous sodium carbonate solution, dried over sodium sulfate and evaporated.

The residue was a mixture, mainly of p<->chlorothiophenol and the desired thioether, 1-(p-chlorophenylthiomethyl)-1-decarboxylasaloside, which was chromatographed with benzene/ethyl acetate (2:1) as eluent. The fractions containing the product (thin layer chromatography) were combined and, on evaporation, gave a colorless solid foam which liquefied at 42-46°C.

Example 5

1.4 g of 1-(diethylaminomethyl)-1-decarboxylasaloside, 2 ml of cyclohexyl mercaptan and 2 ml of triethylamine were heated in 50 ml of ethanol at reflux. By means of the method described in example 4, including the addition of further cyclohexyl mercaptan and triethylamine after 18 hours, the product, 1-(cyclohexyl-thiomethyl)-1-decarboxylasalocid, was obtained as a colorless solid foam after purification on a silica gel column.

Example 6

4v5 g of 1-(diethylaminomethyl)-1-decarboxylasaloside, 5 ml of benzyl mercaptan and 5 ml of triethylamine were heated under reflux overnight in 100 ml of ethanol. After cooling and evaporation of the solvent under reduced pressure, the residue was chromatographed on silica gel with hexane/ethyl acetate (5:1). The fractions containing the product were combined and evaporated. The product thus obtained was contaminated by small amounts of benzyl mercaptan and was purified by preparative layer chromatography on 6 20 x 20 cm silica gel plates with hexane/ethyl acetate (4:1). The pure product, 1-(benzylthiomethyl-1-decarboxylasaloside), was obtained as a colorless solid foam.

Example 7

A solution of 3 g of the Mannich base prepared according to example 2, 3 ml of mercaptoacetic acid and 3 ml of triethylamine in 100 ml of ethanol was

heated 36 hours at reflux. The ethanol was then removed under reduced pressure and the residue in methylene chloride solution washed with dilute aqueous sodium carbonate solution, water, 0.5N hydrochloric acid and water. After drying and evaporation, the crude product was chromatographed on silica gel with 4% methanol in chloroform. As the product was found to absorb sodium ions from inorganic material, the eluate containing the product was washed before evaporation with highly dilute hydrochloric acid. 1-[(carboxymethyl)thiomethyl]-1-decarboxylasaloside was thus obtained as a colorless foam.

Example 8

A solution of 1.85 g of 1-(diethylaminomethyl)-1-decarboxylasaloside | and 2 ral of triethylamine in 50 ml of ethanol were heated for 32 hours at reflux while a slow stream of hydrogen sulphide was passed through the solution. After evaporation of the solvent under reduced pressure, the residue was chromatographed on preparative layer plates (20 x 20 cm, silica gel). The product, 1-mercaptomethyl-1-decarboxylasaloside, was obtained as a colorless solid foam.

Example 9

~A solution of 1 g of 1-(diethylaminomethyl)-1-decarboxylasalocid and

2 ml of triethylamine in 50 ml of ethanol were heated for 8 hours at reflux whereby gaseous methyl mercaptan was introduced. After evaporation and purification by preparative layer chromatography, 300 mg of 1-(methylthiomethyl)-1-decarboxylasalocid was obtained as a colorless solid foam.

Example 10

A solution of 1.4 g of 1-(diethylaminomethyl)-1-decarboxylasaloside,

2 ml of furfuryl mercaptan and 2 ml of diethylamine in 50 ml of ethanol were heated for 42 hours at reflux whereby 2 ml of additional base and 2 ml of mercaptan were added after 18 hours. After evaporation.and

purification by preparative layer chromatography yielded the product, 1-(furylmethylthiomethyl)-1-decarboxylasalocid, as a colorless solid foam.

Example 11

By the process described above and using the appropriate thiol, the compound of formula I can be prepared in which R is allyl, 2-aminophenyl, 4-aminophenyl, 4-bromo-3-methylphenyl, 4-bromophenyl, t-butyl, 2-butyl, tert.butyl, isobutyl, carboethoxy-methyl, carbomethoxymethyl, 2-carboxyethyl, o-carboxyphenyl, 4-chloro-benzyl, 2-n-decylaminoethyl, 2,5-dichlorophenyl, 3,4-dichloropheny1, 2-diethylaminoethyl, 2- diisopropylaminoethyl, 1-dodecyl, ethyl, 4-fluoro-phenyl, n-heptyl, n-hexadecy1, 2-hydroxyethyl1, 2-ethoxyethyl, 2-ethylthioethyl, 1-methyl1-2-imidazolyl, 2,3-dihydroxypropyl, 2- pyridyl, 4-pyridyl, 3-hydroxy-2-pyridy1, 2-thiazolyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxypheny1, 2-methyl-2-butyl, 3-methyl-l-buty1, 4-nitrophenyl, 1-nonyl, 1-octyl, pentachlorophenyl1, pentafluorophenyl1, 1-pentyl, 3-phenyl-1-propyl, 1-propyl, 2-propyl, 2-quinolyl, 2 , 3 ,5 ,6-tetrafluorophenyl, p- tolyl, 2, 4, 5-trichlorophenyl, 7-trifluoro-

Claims (13)

1. Procedure for the preparation of compounds with the formula where R"*" means aryl, substituted aryl, heteroaryl, aralkyl, cycloalkyl, lower alkyl, lower alkenyl, caralkoxy-lower alkyl, aminoalkyl, lower alkyl1-aminoalkyl1, di(lower-alkyl)-aminoalkyl, hydroxyalkyl, alkoxyalkyl1, thioalkyl , carboxy-lower alkyl or hydrogen, characterized by reacting a compound with the form in 2 3 where R and R are lower alkyl or together with nitrogen- The ^ atom forms a 5- or 6-membered heterocyclic ring which may contain a further heteroatom, i' with a compound of formula R^SH, where R^" has the above-mentioned meaning, in the presence of a weak base. 2. Process according to claim 1, characterized in that R"*" is aryl, substituted aryl, aralkyl, cycloalkyl, lower alkyl, carboxy-lower alkyl or hydrogen. 3. Method according to claim 1 or 2, characterized in that R<1> is aryl. 4. Method according to claim 3, characterized in that R"1" is phenyl. 5. Process according to claim 1 or 2, characterized in that R"<*>" is chlorophenyl, cyclohexyl, benzyl or carboxymethyl. 6. Procedure for the manufacture of pharmaceutical preparations containing a. compound of formula I according to the definition in claim 1, characterized by mixing a compound of formula I with non-toxic, therapeutically tolerable solid or liquid carriers and/or excipients common in such preparations. 7. Pharmaceutical preparations characterized by a content of a compound of formula I according to the definition in claim 1. 8. Compounds with formula where R<2> and R<3> are lower alkyl or together with nitrogen- the atom forms a 5- or 6-membered heterocyclic ring which • may contain a further heteroatom. 9. Connection with formula where R<1>means aryl, substituted aryl, heteroaryl, aralkyl, cycloalkyl, lower alkyl, lower alkenyl, caralkyloxy-lower alkyl, aminoalkyl, lower alkylaminoalkyl1, di(lower alkyl)aminoalkyl, hydroxyalkyl1, alkoxyalkyl1, thioalkylalkyl, carboxy -lower alkyl or hydrogen 10. Compounds according to claim 9, characterized in that R<1> is aryl, substituted aryl, aralkyl, cycloalkyl, lower alkyl, carboxy-lower alkyl or hydrogen. 11. Compounds according to claim 9 or 10, characterized in that R 1 is aryl. 12. 1-(Phenylthiomethyl)-1-decarboxylasaloside. 13. Compounds according to claim 9, characterized in that R 1 is chlorophenyl, cyclohexyl, benzyl or carboxymethyl.