SE174792C1 - - Google Patents

Description

Inventor: C E Holmlund ° eh W W Andres Priority Begired Trained March 5, 1958 (United States of America) Present Invention A new process is emerging for the manufacture of antibiotic agents and the associated cyclinic series.

The distinguishing feature of the process is that a tetracycline antibiotic, which lacks a hydroxy group in the 12a position, is oxidized in and of itself to introduce a 12a-thydroxy group. 12α-deoxytetracyclines form the same formations of the tetracyldine series. 12α-deoxytetracycline is produced by chemical reduction of tetracycline with metallic zinc in a surface solution of ammonia. The ammonium lacquer used is Himpligen consisting of a -% - ig It was found necessary that any other aqueous ammonia as a solvent medium, since Other solvents, such as weak & lira media, either do not assist in the reduction of tetracycline liii 12adeoxytetracycline or involve the formation of other, and illegal reduction products. The reaction can be carried out Add a temperature between about 10 and 50 ° C but is carried out at about room temperature, i.e. Indian about 20 Doh 25 ° C. The reaction time is not illegally critical and can generally amount to between about 1 and about 4 hours. A concentration of 4% (w / v) of the tetracycline antibiotic in the aqueous ammonia satisfies, satisfying the reaction. The anyanda zinc bot is aptly available in finely divided form, e.g. such as zinc dust, and the metal should be used in an amount of at least 2 parts by weight per part by weight of tetracycline antibiotic. That in general, a joke necessary • that anyanda a metal amount Over kids 4 parts by weight.

The conditions for the oxidation reaction which according to the invention challenge are relatively critical in the sense that the pH value, the concentration lay, the oxidizing agent, the medium and the temperature affect both the yield of the final product, and. the reaction rate. It has been found that hydroxylation of 12α-deoxytetracycline can be carried out by a pH range between about 2 and 7, and suitably by a pH value of about 4.2 and 4.4 for optimal results. The pH value is suitably adjusted to the desired level, using a suitable buffering agent, e.g. citrate-phosphate-hued, even mu, it ieke, is critical to use 'just, this buffer, .eftersoin, its, only funk. tion, fir to ine, give a pH control.

The reaction can be carried out at a temperature unellan, un.gefar 0 loch un, gefar 500 C and is carried out steam-at ung-efar frame temperature, i.e. between about 25 and 30 ° C.

Examples of lamps are oxidizing agents which can be used in carrying out the described 12α-hydroxylation, such as potassium permanganate, potassium dichromate, potassium ferricyanide, sodium nitrite, potassium nitrite, potassium iodide, iodine and potassium persulfate. Sodium hypo, chlorochlorite and potassium iodate also have anyants but are not suitable, since the action, the rate is much lower and a relatively long time required for the desired hydroxylation. The invention is, however, not the oxidizing agent specified above, but other such agents may also be used, if desired. The amount of oxidizing agent required in the desired hydroxylation can vary in a wide, undurated form. It has generally been found that the oxidizing agent can be present in the reaction mixture in an amount of about 1/8 to about 2 times the molar concentration of the 12α-deoxytetracycline.

The invention, ar lake pa. something, was limited to .ovan describe hydroxylation ay 1.2ad, eoxitetracycline, without ‘kan; as angiyits Even applied to. , hydroxylation: ax other 12α-deoxytetracyclines, I. ex. 7-chloro-6-demethyltetra-2--cycline, 6-deoxytetracycline, 6-demethyltetracycline and 6-demethyl-6-deoxytetracycline. In addition, the described hydroxylation with good results can be applied to 12α-cleoxide dimethylaminotetracycline to form dedimethylaminotetracycline. The starting compound, 12α-deoxide dimethylaminotetracycline, can be prepared by contacting all tetracycline with metallic zinc in weakly acidic medium, e.g. in glacial acetic acid, for at least 72 hours. This reaction results in the elimination of the hydroxyl group in the 12α-position in the tetracycline nucleus and in the elimination of the dimethylamino group in the 4-position in the tetracycline nucleus.

The resulting compound Ran is named 12α-deoxide dimethylaminotetracyldin. This reaction using -chlorotetracycline as starting material and resulting in 12-oxide dimethylaminochlorotetracycline is described in J. A. C. S 76, p. 3574 (1954).

The invention is further exemplified by the following methods, which are set forth in degrees Celsius.

Example 1. 2 g of tetracycline hydrochloride are added to 50 ml of aqueous 15% ammonium hydroxide. 4 g of zinc dust are added: and the mixture is stirred for 2 hours. The excess zinc is filtered off and the filtrate is treated with concentrated hydrochloric acid until the pH is 7. The rap product is filtered off and suspended in 500 ml of water and brought to a solution at pH 1.5. The solution is clarified over diatomaceous earth. The pH of the filtrate is then installed at 4.0 to 4.5, a small amount of solid is filtered off and the filtrate is carefully extracted with ether in a liquid-liquid extractor. The ether extract gives 750 mg of crystalline 12-deoxytetracycline, which is recrystallized from N, N-dimethylformamide and methanol.

Example 2. To a 4 liter flask was added 800 ml of citrate-phosphate buffer (pH 4.4), 100 ml of an aqueous solution of sodium nitrite at a concentration of 800 g / ml and 100 ml of an acidified solution of 12α-deoxytetracycline (prepared according to Example 1) at a concentration of 10 mg / ml. The reaction mixture is stirred on a reciprocating shaker for 3 hours. The reaction mixture is then installed at pH 8.5 to 8.6, extracted several times with butanol, the combined butanol extracts are concentrated to a small volume, and the drawn tetracycline is precipitated by the addition of 10 parts by volume of petroleum ether. The tetracycline is freed from most of the constituents: the impurities by partition chromatography: and: subsequent crystallization. The crystalline tetracycline is identified by determination: of the infrared absorption spectrum, ultraviolet absorption spectrum, paper chromatography and by determination of the microbiological activity.

Example 3. 0.5 ml of an aqueous solution of air 12a-deoxytetracycline at a concentration of mg / ml (prepared according to Example 1) is added to 4 ml of an etherate-phosphate buffer prepared at pH 4.4. 0.5 ml of a solution of potassium permanganate at a concentration of 460 pg / ul was added and the mixture was stirred for 2 hours at a temperature of 28 °. A prom of the mixture is analyzed, and turns out to be essentially free Iran 12a deoxytetracycline. The reaction mixture is tested against Staphylococcus aurens, it being found that the reaction mixture typically has tetracycline antibacterial activity.

Example 4. The experiment of Example 3 is repeated, but using 0.5 ml of a solution of potassium dichromate with a concentration of 860 pg / ml as oxidizing agent. The reaction is carried out for 2 hours at 28 °. Paper chromatography shows narvaro air tetracycline.

Example 5. The procedure of Example 3 is repeated but using 0.5 ml of a solution of potassium ferric cyanide with a concentration of 960 pg / ml as oxidizing agent. The reaction is carried out for 2 hours at 28 °. P: Appear chromatography shows the presence of tetracycline.

Example 6. The hazard test in Example 3 is repeated, but using 0.5 ml of a solution of lumpersulfate with a concentration of 1580, ag / ml, as oxidizing agent. The reaction is carried out for 2 hours at 28 °. Paper chromatography visor narvaro air tetracycline.

Example 7. The experiment of Example 3 is repeated but using 0.5 .mu.l of an iodine broth with a concentration of 480 ggind as oxidizing agent. The reaction is carried out at 28 ° for 2 hours. Paper chromatography shows the presence of tetracycline.

Example 8. The experiment of Example 3 is repeated, but using 0.5 ml of a solution of potassium nitrite: at the concentration of 1000: pg / ml, as oxidizing agent. The reaction is carried out for 2 hours at 28 °. P: App.er chromatography shows the presence of tetracycline.

Example 9. The experiment of Example 3 is repeated but using: of an equivalent amount of potassium iodate as oxidizing agent. The reaction is carried out as in the previous example, but a longer time is required before the reaction mixture is found to contain tetracycline.

Example 10. The experiment of Example 3 is repeated but using an equivalent amount of sodium hypochlorite as the oxidizing agent. The reaction is carried out as in the foregoing example but requires longer suffering before the reaction mixture shows the presence of tetracycline.

Example 11. 2.5 ml of a methanol thaw of 12α-deoxide dimethylaminotetracycline at a concentration of 1 mg / ml, as: prepared by reacting tetracycline aired metallic zinc in glacial acetic acid for 72 hours, sat-Ms to 1 ml of citrate-phosphate buffer, prepared at pH 4.4. 1.5 ml of an aqueous solution of sodium nitrite at a concentration of 1300 pg / ml are added to the mixture. The mixture is stirred for 2 hours at 28 °. A 12α-hydroxylation of the substrate occurs as evidenced by the Rade bacterial activity in turbidimetric testing of portions of the reaction mixture against Micrococcus pyogenes.

Turbidimetric test time per hour vg / ml as dull methylaminotetracycline 2108 Example 12. 7-Chloro-6-demethyl-12α-deoxytetracycline is prepared by chemical reduction of tetracycline with metallic zinc in an aqueous solution of ammonia. After completion of the reaction, the excess zinc is filtered off and the filtrate is cooled in an ice bath and acidified with concentrated hydrochloric acid. The reaction mixture was diluted with citrate-phosphate buffer (pH 4.4), after which a spectrophotometric test of the solution showed the presence of about 850 ml of the 12α-deoxy product. To a 50 nil volume flask was added 1 nil of this to pH 4.4 buffered substrate solution and 1 ml of a solution containing 52 .mu.l of sodium nitrite. The flask is then shaken on a reciprocating shaker for 2 minutes at 28 °. At this time, a spectrophotometric test shows a decrease in the concentration of 7-chloro-6-demethyl-12α-deolithetracycline from an initial concentration of 430 pg / ml to 45 .mu.g / ml. A microbiological test shows a trace of the antibacterial activity expressed as 7-chloro-6-demethyltetracycline, from an initial value of pN / ml to more than 100 pg / ml.

Example 13. The experiment of the preceding example is repeated but using 6-demethyl-12α-deoxytetracycline such as 12α-deoxytetracycline. The concentration in citrate-phosphate buffered solution is approximately 1100 pg / ml. The sodium nitrite solution used has a concentration of 70 pg / ml. After 2 Hinman reaction, a spectrophotometric test shows a decrease in the concentration of 6-demethyl12a-deoxytetracycline from an initial level of 550 pg / ml to 72 pg / ml. A microbiological test shows an increase in antibacterial activity, expressed as 6-demethyltetracycline, from an initial value of 18 pg / ml to 152 pg / ml.

Example 14. The experiment of Example 12 is repeated but aped using 6,12a-dideoxytetra cyclin. The concentration of this in citric phosphate buffered solution is approximately 650 pg / ml. The sodium nitrite solution used has a concentration of 70 pg / ml. Bit completed reaction shows a spectrophotometric test showing a decrease in the concentration of 6,12a-dideoxytetracycline from an initial value of 3 layers / nd to 78 pg / ml. A microbiological test shows a Ruing, of the antibacterial activity, expressed as 6-deoxytetracycline, from an initial value of 28 pg / ml to 92 pg / ml.

Claims (5)

Patent and practice: 1. A process for the preparation of a medal with antibiotic action and associated tetracycline series, characterized in that a tetracycline antibiotic, which lacks a hydroxy group 12a position, is oxidized on its own edge, in order to introduce a 12-hydroxy group . 2. A process according to claim 1, characterized in that the 12α-hydroxylation is carried out at a pH of about 2 and 7. 3. According to patent specification 1 ci1cr 2, designated a-ray, all, the oxidation is carried out with an oxidizing agent present in the reaction mixture in an entrapped Iran about 1/8 to about 2 times the molar concentration of the 12a-deoxytetracycline. in the reaction mixture. 4. A process according to any one of claims 1-3, characterized in that the oxidation is carried out with sodium nitrite, potassium nitrite, potassium permanganate, potassium dichromate, potassium ferricyanide, potassium iodide, iodine or potassium permulfate. 5. A process according to any one of claims 1-4, characterized in that 12α-deoxytetracycline, 12α-deoxide dimethylaminotetracycline, 7-chloro-6-demethyl-12α-deoxytetracycline, 6-demethyl-12α-deoxytetracycline or 6,12α- dideoxytetracycline. Request publications: Patentskrifter frezn Sverige 166 674. Other publications: Naturwissenschaften, 46 (1959), p. 204. Stoekhohn 1g61, Aungi. Boktr. P. A. Norstedt & SUeer. 510089