NO127049B - - Google Patents

Description

Process for the production of 6-deoxytetracyclines.

The invention relates to the preparation of new compounds of the tetracycline series.

These new connections according to

the invention includes 6-deoxytetracyclines with the general formula:

where Ri is hydrogen or hydroxy and the 4-epimers of said 6-deoxytetracyclines. The invention also encompasses the production of the therapeutically active acidic and basic salts and complex compounds of said 6-deoxytetracyclines and the 4-epimers of

said 6-deoxytetracyclines, e.g. the mineral acid salts, the alkali metal salts and the alkaline earth metal salts, as well as various complex compounds, e.g. such as are formed with salts of aluminium, magnesium and calcium.

The new compounds according to the invention are related to the well-known and widely used broad-spectrum antibiotics, tetracycline and oxytetracycline. The 6-deoxytetracyclines described here differ essentially from the aforementioned antibiotics in that the hydroxy group in the 6-position of the naphthacene ring has been replaced by a hydrogen atom. This change brings about a significant difference with respect to the activity of the resulting compounds, as they appear to be equally effective against certain tetracycline-resistant strains of bacteria. It is very surprising that the 6-deoxytetracyclines according to the invention, especially the 6-deoxytetracycline itself, retain the typical broad-spectrum antibacterial activity of the tetracyclines, especially when you take into account that the anhydrotetracycline, which also lacks a hydroxy group in the 6-position in the naphthacene ring, exhibits a significantly lower antibacterial activity than the parent compound.

An appropriate chemical name for the tetracycline, prepared according to the present invention, is according to the nomenclature used by Chemical Abstracts 4-dimethylamino-1,4,4a,5a,6,11,12a-octa-hydro-3,10 ,12,12α-tetrahydroxy-6-methyl-1,11-dioxo-2-naphthacenecarboxamide. The oxy-tetracycline which is analogous to this has a hydroxy group in the 5-position of the ring and is designated in a similar manner. An appropriate common name for these new compounds is 6-deoxytetracycline, resp. 6-deoxyoxytetracycline which will be used here.

One of the more important advantages that the new compounds produced according to the present invention possess, just over the previously described tetracyclines, is their increased stability in acids and alkali. The acid instability of tetracycline and the alkali instability of chlortetracycline are well known. Chlortetracycline in an aqueous solution with a sodium carbonate buffer at pH 9.85 will lose 50 per cent of its activity within 29.2 min. at 23° C. In contrast, 6-deoxytetracycline does not lose more than 1% of its activity during 24 hours under the same conditions. Tetracycline has a half-life of less than 1 min. in 3 normal hydrochloric acid at 100° C. C-deoxytetracycline, on the other hand, has a half-life of 27 hours under the same conditions. Under the same conditions, 6-deoxytetracycline has a half-life of 45 hours. Tetracycline has a half-life of approx. 6 min. in 0.1 normal sodium hydroxide solution at 100° C. In contrast, 6-deoxytetracycline has a half-life of 9y2 hours under the same conditions and 6-deoxyoxytetracycline has a half-life of loi/g hours under the same conditions. These unexpected properties are very valuable inasmuch as the acid instability of tetracycline and the alkali instability of chlortetracycline have limited or completely precluded the use of these antibiotics for many purposes. As a result of a much better stability of the new 6-deoxytetracyclines, it is possible to produce many pharmaceutical products that could not be produced satisfactorily with the previously known tetracyclines. The increased stability also makes it possible to improve the extraction and purification processes as more drastic pH and temperature conditions can be used without the new compounds being destroyed. As mentioned above, the anti-bacterial activity of the 6-deoxytetracyclines is in many respects quite similar to the anti-bacterial activity of the previously known tetracyclines and thus the new compounds can be administered by the physician in the same way and in approximately the same dosage amounts as with the tetracyclines which is currently in use. The new 6-deoxytetracyclines can furthermore, as they exhibit the typical broad-spectrum antibiotic activity of the previously known tetracyclines, be used in the treatment of various infections caused by both Gram-positive and Gram-negative bacteria, where the treatment of such infections with tetracycline or oxytetracycline is desirable.

The antibacterial spectrum of the new compounds representing the amount required to inhibit the growth of various typical bacteria was determined in a conventional manner by the agar dilution smear technique commonly used in testing new antibiotics. The minimum concentrations that cause inhibition, expressed in gamma per milliliters of 6-deoxytetracycline and 6-deoxyoxytetracycline equally against different test organisms are listed in the following table. For the sake of comparison, the anti-bacterial activity of tetracycline against the same organisms is also listed:

From the foregoing, it will be seen that in many respects the antibacterial spectrum of the new compounds produced according to the invention is very close in parallel with the antibacterial spectrum for tetracycline, but in connection with this, 6-deoxy-tetracycline is effective against certain tetracyclines -resistant strains of bacteria, such as Streptococcus hemolyticus gamma hemolytic, Staphylococcus albus, and Streptococcus hemolyticus, group D. Furthermore, both new compounds are much more effective against streptococcus hemolyticus, beta hemolytic than tetracycline.

The 6-deoxytetracyclines are prepared according to the invention by a rather unique catalytic reduction of the corresponding tetracycline, in a solvent solution in the presence of a substance capable of forming a chelate ring with a peri-dioxygenated hydronaphthalene, i.e. 6 -deoxytetracycline is formed by the catalytic reduction of tetracycline, chlortetracycline or bromotetracycline and 6-deoxytetracycline is formed by the catalytic reduction of oxytetracycline.

The reduction process can be carried out by contacting a solvent solution of the corresponding tetracycline with hydrogen in the presence of a suitable catalyst, such as e.g. finely divided metallic palladium or another metal of the platinum family and bone charcoal or palladium hydroxide on bone charcoal.

Furthermore, according to the invention, a method is provided for removing the hydroxy group from the 6-position of a compound of the tetracycline series, consisting in such a compound being catalytically reduced in a solvent solution in the presence of a substance capable of forming a chelate ring with a peri-dioxygenated hydronaphthalene.

Suitable compounds which possess this property of forming a chelate ring and which can be used with good results in this reduction process are boric acid, boron trihalides such as boron trifluoride, aluminum and magnesium salts, such as aluminum chloride and magnesium acetate. Boric acid or boron trihalides appear to be the most useful compounds with regard to obtaining good yields of the desired products. Typically, boric acid or boron trihalide is present in at least mole for mole amounts. The reduction can be carried out at a temperature varying from 0 to 100° C and preferably from approx. room temperature to approx. 50° C and at hydrogen pressure from approx. to approx. 100 atmospheres.

Suitable inert solvents that can be used in the reaction are various polar solvents such as water, dioxane, glacial acetic acid, 2-ethoxyethanol and ethyl acetate. An one-to-one ratio of water and dimethylformamide has proven to be a particularly good solvent for this reaction. A small amount of perchloric acid is usually added to the solution. A concentration of the catalyst of at least 5% by weight of the corresponding tetracycline is necessary and up to approx. 100 percent by weight. The hydrogenation is usually carried out until 1 mole of hydrogen has been absorbed when tetracycline is the starting material, at which point the rate of absorption tends to decrease. When chlortetracycline is used, 2 moles of hydrogen are naturally required. Some caution must be exercised so that the hydrogenation does not continue for too long, as further and unwanted reactions may thereby take place with the formation of less desirable products.

The substances capable of forming a chelate ring, as described above, which are used in the described reduction process, are very important reagents as they apparently act as complex-forming substances and serve to prevent the reduction of the oxygen functions at 11- and 12- the positions in the naphthalene ring. These reagents are very important in the reduction as in the absence of such agents the 12-position is reduced before the 6-position, and the resulting compound exhibits no biological activity. In the described reduction process, however, the chelation serves to bind these two oxygen groups and prevents their reduction so that the antibacterial activity of the compound is maintained.

After the hydrogenation is complete, the 6-deoxytetracycline or 6-deoxytetracycline is recovered by known means, e.g. by removing the catalyst and concentrating the solution. The product is evaporated to dryness, purified by fractional precipitation in methanol and can be further purified by recrystallization in alcohol in the usual way. The neutral product formed in this way can be converted into a mineral acid salt, e.g. the hydrochloride by treatment with acids, such as hydrochloric acid, at a pH of lower than approx. 4. Other acidic salts, such as the sulfuric acid salt. the phosphoric acid salt, the trichloroacetic acid salt, etc., may be formed in a similar manner. Preferably, the 6-deoxytetracyclines are suspended in a suitable solvent during the acidification. The alkali metal and alkaline earth metal salt can be simply formed by treating the amphoteric compounds with approximately one equivalent of the chosen base, e.g. sodium hydroxide, potassium hydroxide, calcium hydroxide, barium hydroxide, etc. The metal salts can be prepared in aqueous solution or in a suitable solvent. Preferably, the basic salts are prepared at a pH of 6 or higher. The free base can be obtained at a pH within the range of approx. 4-6. The complex compounds, such as The 6-deoxytetracycline ammonium gm-conate complex, can e.g. is formed by simple mixing of the hydrochloride salt of the new compounds and aluminum gluconate in an aqueous solution.

The 4-epimers of the 6-deoxytetracyclines, i.e. which have an epimeric form at the carbon atom at position four, the same as described in connection with other tetracycline compounds, can also be formed by simple regulation of the hydrogen ion concentration of a concentrated solution of the antibiotic within a range of pH 3.0 to 5.0, after which the solution is allowed to stand until the isomerization has reached equilibrium.

The isomerization is most conveniently carried out at room temperature, although a greater rate of conversion takes place at a higher temperature. The pH must be within the range of approx. 3.0 to 5.0, preferably between 3.5 and 4.5. Some epimerization will take place at hydrogen ion concentrations outside these ranges, and even in distilled water, but the rate is very slow. The concentration of the antibiotic in the aqueous solution must be as high as possible to achieve the greatest epimerization rates. A complete equilibrium may require a time period of approx. 24 hours at 25° C, but satisfactory equilibrium may require a significantly shorter time under special conditions. In general, however, the best results are achieved by allowing the solutions to stand for periods of a week or more. An equilibrium seems to be reached in most cases at approx. 50 per cent, i.e. approximately half of the antibiotic is converted to epimers at equilibrium.

As the concentration is an important factor in achieving high yields in short periods of time, a solvent system is chosen which gives the highest concentration of the antibiotic. These solvent systems can be treated with buffer substances to obtain a pH within the preferred range. Various solvents include methanol, ethanol, butanol, acetone, 2-ethoxy-ethanol, 2-methoxy-propanol, glacial acetic acid, tetrahydrofuran, dimethylformamide and mixtures of these solvents. Other solvents can also be used. A preferred agent as a buffer substance is sodium dihydrogen phosphate, although other buffer substances and buffer substance pairs can be used which will keep the hydrogen ion concentration within the desired range.

In the following, the invention will be described in more detail in connection with the listed examples.

Example 1.

12 grams of tetracycline hydrochloride are suspended in 10 parts by volume of a 1 to 1 mixture of water and dimethylformamide. 1.48 g of boric acid, 12 g of unreduced 5% palladium on charcoal and 0.5 ml of perchloric acid are added to the mixture. The hydrogenation is carried out by reaction with hydrogen at approx. 2.8 kg/cm- on a Parr shaker for approx. 2½ hours. The hydrogenation is completed after approx. 1 mole has been absorbed. The solution is filtered and the catalyst is washed with 10 ml of dimethylformamide and then another washing is carried out with 10 ml of water.

Example 2.

The filtrate obtained by the method described in example 1 is adjusted to pH 3.0 with concentrated ammonium hydroxide and evaporated to dryness in a vacuum.

50 ml of water-saturated butanol is added, and

the mixture is stirred and filtered. The activity is concentrated in water, and the pH is adjusted to 3.0, and then filtered. The filtrate is adjusted to pH 1.5 and back-extracted with 100 ml of butanol. The butanol extract is concentrated to approx. 5-10 ml in an atmosphere of nitrogen. The solution is infected and left for 18 hours at room temperature. The crystals are filtered, washed with acetone and then with ether and dried in vacuum at 40° C. for 20 hours so that 179 mg of 6-deoxytetracycline is obtained.

Example 3.

Two-thirds of a gram of 6-deoxytetracycline is suspended in 13.5 ml of ethyl alcohol, 0.5 ml of concentrated hydrochloric acid is added to adjust the pH to 0.8 to 1.0. The solution remains for 3y2 hours. The 6-deoxytetracycline hydrochloride crystals that form are dried in vacuum at 100° C. for 20 hours.

Analysis: Calculated for C^H-rJS^ClO-,

: C, 56.7; H, 5.38; N, 6.02; Cl,

7.69; 0, 24.1;

Found: C, 56.52; H, 5.46; N, 5.94; Cl, 7.71;

0, 23.89.

The product has an optical rotation of [a] ^ = -292° in o.lN H^SCm.

The ultraviolet absorption spectrum is determined from a sample of the compound in 0.1 N H2SO4 at a concentration of 30.65 gamma per milliliters.

The infrared absorption spectrum is determined from a sample of the compound, mixed with crystals of KB r and pressed into a disk.

Example 4.

The procedure according to example 1 is repeated with the only exception that 12 g of oxytetracycline is used as starting material instead of the tetracycline hydrochloride.

Example 5.

To the reduction solution obtained in example 4, 5 g of diatomaceous earth (Hyflo) is added and the mixture is filtered. The filter cake is washed with 50 ml of a 50-50 mixture of dimethylformamide and water. The filtrate and washing liquids are combined, so that a volume of 163 ml is obtained. The pH of the combined washing and filtrate liquids is adjusted to 3.0 with concentrated ammonium hydroxide, and the solution is evaporated to dryness in a vacuum at 40-60° C. The solids are extracted with 75 ml of water for one hour while stirring. The sludge is filtered. The pH of the solution is adjusted to 1.0 with hydrochloric acid. The active substance is extracted twice in butanol. The butanol extracts are combined and evaporated to dryness. 7 ml of acetone is added to the dried butanol extract, and hydrochloric acid is added to adjust the pH to 1.0. The solids are removed by centrifugation. The supernatant liquid is strained, and the crystals that form are allowed to stand for 6 hours, filtered and washed with acetone and then in ether. The crystals are dried in a vacuum at 40° C. for 12 hours. Yield 84.7 mg of 6-deoxyoxytetracycline.

Analysis: Calculated for C-HwN-ClOs,

C, 54.90; H, 5.20; N, 5.83; Cl, 7.38;

O, 26.70;

Found: C, 54.48, H, 4.81; N, 5.50; Cl, 7.74;

O, 26.55;

The product has an optical rotation of [a] £ = —251° in 0.1 N HjSO-i.

The ultraviolet absorption spectrum is determined from a sample of the compound in 0.1 N H2SO4 at a concentration of 40.76 gamma per milliliters.

The infrared absorption spectrum is determined from a sample of the compound mixed with crystals of KBr and pressed into a disk.

Example 6.

To 1.0 g of tetracycline hydrochloride is added 25 ml of a one-to-one mixture of dimethylformamide and water. 3 ml (10 mol equivalents) of 45% boron trifluoride ether are added, and the pH is adjusted to 1.3 with 2 ml of triethylamine. 1.0 g 5% palladium on carbon is added, and the mixture is placed on a Parr shaker, and allowed to react with hydrogen for 100 min. (hydrogen absorption is one mole). The mixture is filtered, and the insoluble matter is washed with 10 ml of water. There, 6-deoxytetracycline is formed.

Example 7.

25 ml of a 1 to 5 mixture of dimethylformamide and water is added to 2.5 g of tetracycline hydrochloride. 1.1 g of magnesium acetate (Mg(C2H:(02)2.4H20) is added. The pH is adjusted to 1.8 with hydrochloric acid. 2.5 g of 5% palladium on charcoal and 2 drops of perchloric acid are added, and the mixture is placed on a Parr shaker, and allowed to react with hydrogen for 71 min. (Hydrogen uptake is 1 mol). The reduced solution is filtered, and the insoluble is washed with 0.1 N hydrochloric acid. 6-deoxytetracycline is formed.

Example 8.

25 ml of a 1 to 1 mixture of dimethylformamide and water is added to 2.5 g of tetracycline hydrochloride. The pH is adjusted to 1.8 with hydrochloric acid, and 0.55 g of calcium chloride, 2 drops of perchloric acid and 2.5 g of 5% palladium on carbon are added. The mixture is placed on a Parr shaker and allowed to react with hydrogen for 97 min. (absorption of 1 mol Hj). The mixture is filtered and washed with 5 ml N hydrochloric acid. There, 6-deoxytetracycline is formed.

Example 9.

To 1 g of tetracycline hydrochloride is added 25 ml of a 1 to 1 mixture of dimethylformamide and water, which contains 0.27 g of aluminum chloride. The pH is adjusted to 1.5 with perchloric acid. 1.0 g of 5% palladium on carbon is added and the mixture is placed on a Parr shaker for 150 min.

(Absorption of 1 mol H2). The mixture is filtered. There, 6-deoxytetracycline is formed.

Example 10.

1 ml of glacial acetic acid is added to 5 mg of 6-deoxytetracycline. The mixture is shaken and allowed to equilibrate at room temperature for 18 hours and then filtered. Paper strip chromatography shows the presence of 6-deoxy-4-epitetracycline.

Example 11.

1 ml of glacial acetic acid is added to 5 mg of 6-deoxyoxytetracycline. The mixture is shaken and left to equilibrate at room temperature for 18 hours and then filtered. Paper - strip chromatography shows the presence of 6-deoxy-4-epioxytetracycline.

Example 12.

To 1.0 g of tetracycline hydrochloride is added 0.13 g of boric acid dissolved in 28 ml of a 1 to 1 mixture of dimethylformamide water. The pH is adjusted to 2.1 with hydrochloric acid. To 13 ml of this solution is added 0.75 g of 5% palladium on charcoal. The mixture is placed in a stainless steel bomb and allowed to react with hydrogen at a pressure of 134 kg/cm- for 80 min. The mixture is filtered and the insoluble matter is washed with water. Spectrophotometric examinations of the reduction filtrate show the presence of 6-deoxytetracycline.

Example 13.

The procedure according to example 1 is used with the exception that chlortetracycline hydrochloride is used as starting material instead of the tetracycline hydrochloride. The chlortetracycline hydrochloride is added to 15 ml of a 50:50 mixture of dimethylformamide and water and the hydrogenation is carried out as in example 1 until 2 moles of hydrogen have been absorbed. Chromatographic examination of the product shows the presence of 6-deoxytetracycline.

Example 14.

To 4 ml of a solvent solution prepared by mixing 25 ml of dimethylformamide, 25 ml of water, 32.5 mg of boric acid and one drop of perchloric acid is added 5.67 mg of 4-epioxytetracycline neutral (J.A.C.S. 79, 2849 (1957)) . 7 mg of 5% palladium on carbon is added, and the mixture is brought into contact with hydrogen at a pressure of 2.1 kg/cm- for 6 hours. Paper chromatographic and spectrophotometric analyzes show the presence of 6-deoxy-4-epioxytetracycline.

Example 15.

To 4 ml of a solvent solution, prepared by mixing 25 ml of dimethylformamide, 25 ml of water, 32.5 mg of boric acid and one drop of perchloric acid, is added 5.0 mg of 4-epibromotetracycline ammonium salt (J.A. C.S 79, 2849 (1957) ). 7 mg of 5% palladium on carbon is added, and the mixture is brought into contact with hydrogen at a pressure of 2.1 kg/cm- for 6 hours. Paper chromatography and spectrophotometric analyzes show the presence of 6-deoxy-4-epitetracycline.

Example 16.

To 4 ml of a solvent solution prepared by mixing 25 ml of dimethylformamide, 25 ml of water, 32.5 mg of boric acid and one drop of perchloric acid, is added 5.0 mg of 4-epichlorotetracycline hydrochloride (J. A.C.S. 79, 2849 (1957)). 7 mg of 5% palladium on carbon is added, and the mixture is brought into contact with hydrogen at a pressure of 2.1 kg/cm - for 7 hours. Paper chromatography and spectrophotometric analyzes show the presence of 6-deoxy-4-epitetracycline.

Example 17.

To 4 ml of a solution prepared by mixing 25 ml of dimethylformamide, 25 ml of water, 32.5 mg of boric acid and one drop of perchloric acid, 6.0 mg of bromotetracycline is added. 7 mg of 5% palladium on carbon is added, and the mixture is brought into contact with hydrogen at 2.1 kg/cm - for 6 hours. Paper chromatographic and spectrophotometric analysis shows the presence of 6-deoxytetracycline.

Example 18.

To 4 ml of a solution, prepared by mixing 25 ml of dimethylformamide, 25 ml of water, 32.5 mg of boric acid and one drop of perchloric acid, is added 5.0 mg of 4-epitetracycline (J.A.C.S. 79, 2849, (1957)). 7 mg of 5% palladium on carbon is added, and the mixture is brought into contact with hydrogen

at 2.1 kg/cm<2> for 4 hours. Paper chromatographic and spectrophotometric analysis shows

the presence of 6-deoxy-4-epi-tetracycline.

Claims (5)

Procedure for preparing 6-de-Doxytetracyclines of the formula: where Rl is hydrogen or hydroxy and salts and complex compounds thereof, characterized in that a solution of tetracycline, chlortetracycline, bromotetracycline or oxytetracycline or the 4-epimer of such a compound in a polar inert solvent is catalytically hydrogenated, preferably with the aid of a catalyst of the platinum group on bone charcoal, in the presence of a substance capable of forming a chelate ring with a peri-dioxygenated hydronaphthalene, if desired the resulting 6-deoxytetracycline is brought into solution to react with an acid or base or with a complexing compound . 2. Method as stated in claim 1, characterized in that the catalyst concentration is 1 at least 5% by weight of the tetracycline used. 3. Method as stated in claims 1-2, characterized in that the reduction is continued until approximately 1 mol hydrogen has been absorbed for each mole of tetracycline or oxytetracycline or about 2 moles for each mole of chlortetracycline or bromotetracycline. 4. Method as stated in any one of claims 1 to 3, characterized in that said substance capable of forming a chelate ring is boric acid or a boron trihalide. 5. Modification of the method according to claim 1 for the production of 4-epimers of 6-deoxytetracyclines with the general formula I, characterized in that a concentrated solution of said 6-deoxytetracycline is adjusted to a pH of about 3.0 to 5, 0, preferably 3.5-4.5, and allow the solution to stand until the isomer ice has reached equilibrium.