NO131589B - - Google Patents

Description

Process for the production of biologically active stable tetracycline compounds.

The present invention relates to a method for the production of biologically active, stable, substituted 6-deoxy I

tetracyclines which are represented by the following general formula:

and acid addition salts thereof, where Rj

or R2 means nitro, and the other is hydrogen, nitro, amino or halogen, R., is hydrogen or methyl and R, is hydrogen or hydroxy, and these compounds are prepared by a compound of the general formula I, where Rx and/ or R2 is hydrogen, or an acidic addition juice thereof, is brought to react with a nitrating agent and then, if desired, a reduction of the nitro group to an amino group is carried out, and if R and R2 in the starting material were both hydrogen, the amino compound formed is possibly brought to to react with a nitrating agent.

Certain of the new compounds according to this invention, e.g. nitrotetracyclines, are produced by reaction of 6-demethyl-6-deoxytetracycline, 6-deoxytetracycline or 5-hydroxy-6-deoxytetracycline with potassium nitrate, preferably a molar equivalent thereof, and a strong acid, e.g. sulfuric acid, at a temperature varying from approx. ^-15° C to approx. + 15° C. The resulting nitrotetracycline is usually isolated from the reaction mixture as the acid sulfate by precipitation with cold ether. The free base can be obtained by regulating an aqueous solution of the product to a pH of from oa. 4 to 6 with a mild alkali, ie sodium carbonate. The resulting product can be called nitro-6-deoxytetracycline, nitro-6-demethyl-6-deoxytetracycline or nitro-5-hydroxy-6-deoxytetracycline, depending on the starting material used.

When 6-demethyl-6-deoxytetracycline is used as starting material, it has been shown that the resulting nitration product is a mixture which can be separated into two specific compounds by fractional crystallization. For convenience, these are designated as nitri-6-demethyl-6-deoxytetracycline isomer (A) and nitro-6-demethyl-6-deoxytetracycline isomer (B).

The isolation of nitro-6-demethyl-6-deoxytetracycline (B) from the crude nitration product is effected by fractional crystallization. A rough separation of inorganic and organic compounds is carried out with the crude ether-precipitated nitration product using ethanol as solvent. The organic product is then split into two components using the solubility differences in methanol of the resp. free bases. Nitro-6-demethyl-6-deoxytetracycline (B) is isolated as a crystalline sulfate salt from the methanol solution and purified by forming the free base and then recrystallizing the free base in the usual way. The two compounds are distinguished from each other by spectral data and biological activity. The main difference in the ultraviolet spectra between nitro-6-demethyl-6-deoxytetracycline (A) and nitro-6-demethyl-6-deoxytetracycline (B) is significant in the range of long wavelengths. In 0.1N HCl the absorption maxima of nitro-6-demethyl-6-deoxytetracycline (B) is at 350 mp., while nitro-6-demethyl-6-deoxytetracycline (A) absorbs at 360 m\ in. In 0.1M Na2Bi07 nitro-6-demethyl-6-deoxytetracycline (B) has a well-defined absorption maximum at 370 m^, while nitro-6-demethyl-6-deoxytetracycline (A) has a very broad absorption maximum showing a weak peak at 350 m\ x.

The infrared absorption spectra of nitro-6-demethyl-6-deoxytetracycline (B) exhibit nitro absorption bands at 6.54 myi and 7.46 m(.i, essentially the same as the nitro absorption bands of nitro-6-demethyl- 6-deoxytetracycline (A) The complete infrared curve for nitro-6-demethyl-6-deoxytetracycline (B) is, however, otherwise different from the infrared curve for nitro-6-demethyl-6-deoxytetracycline (A).

The structure of nitro-6-demethyl-6-deoxytetracycline (A) and nitro-6-demethyl-6-deoxytetracycline (B) has not yet been proven with certainty, but it is believed that the two compounds are positional isomers with the nitro group attached to the aromatic ring in the tetracycline nucleus in the ortho position to the hydroxyl in one compound and in the para position to the hydroxyl in the other compound.

The nitrotetracyclines produced in this way can be reduced either chemically or catalytically to form the aminotetracyclines. The catalytic reduction can be carried out in a polar solvent such as water, a lower alkanol, e.g. methanol, ethanol, etc., a lower alkoxy-lower alkanol, e.g. 2-methoxyethanol or 2-ethoxyethanol, or a lower alkanoic acid, e.g. acetic acid or propionic acid in a mineral acid solution, e.g. hydrochloric acid or sulfuric acid, and in the presence of a noble metal catalyst, such as finely divided palladium, rhodium or another metal in the platinum family. The pure metal can be used or the metal can be used in the form of an oxide or hydroxide and preferably the catalyst is suspended on one of the usual carriers such as finely divided aluminum oxide, activated bone charcoal, diatomaceous earth etc. The reduction can be carried out at temperatures from approx. 10° C to approx. 40° C, and preferably at room temperature, i.e. at approx. 25° C and a hydrogen pressure of approx. 1 to approx.

3 atm.

The aminotetracycline thus prepared can be recovered from the reaction mixture by any desired means, such as removal of the catalyst and concentration of the solution. The product is evaporated to dryness and purified aminotetracycline is obtained by precipitation from ethanol-ethyl acetate. The product can be purified further if desired by recrystallization in alcohol in the usual way.

The nitrotetracyclines can also be reduced to aminotetracyclines by a chemical reduction process where the nitrotetracycline is brought into contact with a hydrogen-producing compound such as metallic zinc in a mildly acidic medium such as hydrochloric acid, acetic acid, etc., at a temperature of from 10 to 40°C and for a time of from 15 minutes to approx. 2 hours. The concentration of the nitrotetracycline in the acidic medium depends on its solubility. The zinc used for the reaction should preferably be in finely divided form, e.g. zinc dust and this material must be used in a quantity of approx. 0.35 parts by weight of the metal per weight of the nitrotetracycline. Amounts of metal greater than approx. 5 parts by weight are generally not necessary. The reduced solution contains the desired aminotetracycline which can be recovered from the solution in the usual way.

The aminotetracyclines can also be nitrated under the indicated conditions to form mono-substituted nitrotetracycline, to form an amino-nitro-6-deoxytetracycline.

The new tetracyclines according to the invention are amphoteric compounds and consequently acid addition salts can easily be prepared, i.e. both mono- and di-salts (when an aminotetracycline is used). Generally, the preferred mineral acids are hydrochloric acid, sulfuric acid, phosphoric acid and the like, although organic acids such as trichloroacetic acid can also be used. The acid addition salts of the new tetracyclines can be prepared by treating the amphoteric compound with about two equivalents or more of the selected acid. Preferably, the tetracycline is suspended in a suitable solvent during the acidification.

The aminotetracyclines which are formed either by the previously described catalytic reduction processes or chemical reduction processes, can be diazotized by reaction with nitric acid, so that the diazonium salt is formed. The diazonium group can then be replaced using the classical method with hydrogen, hydroxy, alkoxy, halogen (Sandmeyer reaction) cyan, etc. If desired, the diazonium salt can be coupled with phenols and tertiary amines in weakly acidic, neutral or alkaline solution to form strongly colored azo compounds .

The new tetracyclines according to up-

invention are biologically active and exhibit a broad-spectrum antibacterial activity like the previously known tetracyclines. The antibacterial spectrum of certain of these compounds, representing the amount required to prevent or inhibit the growth of various typical bacteria, was determined in the usual manner by the agar dilution smear plate technique commonly used in the investigation of antibiotics. The minimum concentrations expressed in gamma per millimetres, to prevent or inhibit the growth of different test organisms, are listed in the following table. For the sake of comparison, the antibacterial activity of tetracycline against the same organisms is also listed.

The new tetracycline compounds according to the invention are significantly more stable against acid and alkali than the parent compound tetracycline. Moreover, the aminotetracyclines are much more soluble than the parent compound.

In the following, the invention will be described in more detail in connection with a number of examples.

Example 1.

Preparation of crude nitration product of

6- demethyl- 6- deoxytetracycline.

To a solution of 900 mg (2 mg mol) 6-demethyl-6-deoxytetracycline hydro-

chloride [J. A. C. S. 80, 5324 (1958)] prepared by bringing a polar solvent solution of 6-demethyltetracycline [J. A. C. S. 79, 4561 (1957)] in contact with hydrogen in the presence of boric acid and a noble metal catalyst until approximately 1 mole of hydrogen had been absorbed for each mole of 6-demethyl-tetracycline in 50 ml of concentrated H2SO4 at 0° C., was added 200 mg ( 2 m mol) KNOs. The light brown reaction solution was stirred at 0° C. for 20 minutes. The cold solution was gradually poured into 1 liter of diethyl ether at 5° C. The addition was carried out at such a rate that the temperature of the ether remained between 5° and 10° C. The light yellow solid which immediately precipitated was deposited gradually i

the ether solution. Most of the ether solution was decanted and the solid filtered, washed with cold ether (4 x 10 ml.), and dried in vacuo at room temperature for 4 hours. Weight of solid: 1.01 g.

200 mg of the above nitration product was dissolved in 5 ml of H 2 O with stirring. The light brown solution (PH 1.3) was adjusted to pH 5.0 with 2N Na 2 CO 3 . A brownish-yellow solid precipitated. This mixture was allowed to stand with stirring in an ice bath for 1 hour. The solid (as the free base) was filtered, washed with H 2 O (3x5 ml.) and dried in vacuo at 60° C. for 4 hours. Weight 80 mg. Melting point, chars at 197° C.

Analysis:

Calculated for C21H21N3O9.H2O (494.4):

50.9 percent C; 5.08 percent H; 8.46 percent N;

7.26 percent H2O.

Found: 50.63 percent C; 4.84 per cent. H; 8.79 percent N;

5.29 percent HjO (loss on drying).

The ultraviolet spectrum of this product has changed character from the spectrum of the starting material 6-demethyl-6-deoxytetracycline. In 0.1 N hydrochloric acid there is a 5 m(x reduction in the short wavelength deabsorption to 262 m.i, while in the long wavelength absorption there is a 10 m.in increase to 355 m[i. The infrared spectrum which is also different from the spectrum of 6-demethyl-6-deoxytetracycline, has nitro group absorption bands at 6.56 ^ and 7.45 n.

Example 2.

Separation of crude nitration product into nitro-6-demethyl-6-deoxytetracycline (A) and nitro-6-demethyl-G-deoxytetracycline (B).

A slurry of 11.78 g of the ether-precipitated nitration product of 6-demethyl-6-deoxytetracycline in 500 ml of absolute ethanol was heated to boiling on a steam bath. The insoluble material was immediately filtered, washed with hot absolute ethanol (2 x 25 ml) and then with ether (3 x 50 ml). The solid was air dried at room temperature. Weight 3.70 g. The cloudy ethanol filtrate and ethanol washings were combined and evaporated under vacuum to dryness at 30° C. Weight 8.1 g.

The solid residue from the ethanol evaporation was slurried in 300 ml of ethanol (pH in H2O 2.5) and then triethylamine was added until pH 5.0 (in H2O) was reached. An insoluble dark yellow crystalline solid was filtered, washed with methanol (2 x 5 mL), and dried under vacuum at 60°C for 1 hour to give 2.78 g of nitro-6-demethyl-6-deoxytetracycline (A). The methanol filtrate and washings were combined and adjusted to pH 1.8 with 6 N H 2 SO 4 . After cooling the solution in an ice bath, a pale yellow crystalline solid (needle-like clumps) began to precipitate. The mixture was stirred in an ice bath for two hours and then the solid was filtered, washed with ether (5 x 10 ml) and dried under vacuum at 60°C for one hour to give 1.8 g of nitro-6-demethyl -6-deoxytetracycline (B).

Example 3.

Preparation of the free base of nitro-6-demethyl-6-deoxytetracycline (B)

500 mg of the sulfate salt of nitro-6-demethyl-6-deoxytetracycline was stirred for 20

ml. water. A small amount of an insoluble solid was filtered through a filter -

aid on a sintered glass funnel. The

clear yellow filtrate (pH 1.7) was adjusted to pH 5.2 with 2 N Na 2 CO 3 . A yellow, partially crystalline solid was precipitated. The mixture was heated to approx. 80° C in a steam bath and then cooled in an ice bath. The solid still appears to be very fine and remains suspended in solution. The suspension was then frozen in a dry ice methylcellosolve bath. After it had remained frozen for an hour, the mixture was allowed to warm to room temperature. The filtered solid was washed with water (2x5 ml.). The solid was exposed to vacuum at room temperature overnight and then dried at 60°C for one hour. Weight 284 mg.

Example 4.

Recrystallization of the free base of nitro-6-demethyl-6-deoxytetracycline (B).

A mixture of 70 mg of the free base (the product according to Example 3) was heated on a steam bath. A small amount of an insoluble amorphous solid was filtered off while hot. The filtrate which became cloudy during the filtration was reheated to a clear solution. On cooling slowly to room temperature, a yellow crystalline solid (needle-like clumps) precipitates. The solid was filtered, washed with 1 ml of water and dried under vacuum at 60°C for one hour. Weight 37.2 mg.

A portion of this free base was heated in pure toluene in a soxhlet extraction apparatus. The extraction means contained calcium hydride and the yellow toluene solution was heated under reflux for 4 hours. This method was used to remove all water of crystallization from the compound.

A yellow solid was obtained which was heated at 140° C. for two hours under vacuum and then immediately analysed.

Analysis:

Calculated for C21H21N3O9 — 0.2 mol toluene: 56.2% C, 4.70% H, 8.84% N,

30.3 percent O.

Found: 55.94% C, 5.47% H, 8.68% N,

29.73 percent O (direct).

In 0.1N HC1 the ultraviolet absorption maximum is at 350 m|i. In 0.1 M Na 2 B 4 O 7 , the ultraviolet absorption maximum is at 370 mji. The infrared spectrum has nitro group absorption bands at 6.54 |j, and 7.46 \i.

Example 5.

Preparation of the hydrochloride salt of nitro-6-demethyl-6-deoxytetracycline (B).

To a slurry of 250 mg of the free base prepared as indicated in Example 4 in 25 ml of n-butanol; 7 drops of concentrated HC1 were added. A small amount of insoluble solid was filtered and to the clear yellow filtrate was added another 7 drops of concentrated HCl. The solution cooled in an ice bath became slightly cloudy, but no solid precipitated. On heating to 30° C. with some scraping, a pale yellow crystalline solid (needle-like clumps) began to precipitate. After the mixture had stood cold (4° C.) overnight, the solid was filtered, washed with a few drops of n-butanol and dried under vacuum at 100° C. for 3 hours. Weight 223 mg.

Example 6.

Preparation of amino-6-demethyl-6-deoxytetracycline (A). 51 mg of nitro-6-demethyl-6-deoxytetracycline base (A) prepared as indicated in Example 2 was suspended in 5 ml of absolute ethanol. 3 drops of 6 N hydrochloric acid were added to this solution. The solid was gradually dissolved. 5 mg of platinum oxide was added to the clear yellow solution. The mixture was shaken vigorously under a hydrogen atmosphere for 10 minutes and a slight excess of hydrogen just opposite the theoretical volume amount was then absorbed. The solution was filtered and the catalyst was washed twice with 2 ml of absolute ethanol. The filtrate and washings were combined and the combined solution was concentrated to dryness under vacuum. The dark yellow solid residue was dried under vacuum at 60° C. for 1 hour. The yield of the product was 40 mg.

; 0.1N HCl 215 265 34g . 0.1N NaOH 241 27Q 3?7 max r max r

Example 7. I

Preparation of amino-6-demethyl-6-deoxytetracycline (B).

The procedure according to the preceding example was repeated except that nitro-

6-demethyl-6-deoxytetracycline base (B), prepared as in Example 2, was used instead of the A isomer. The time required for the hydration was approximately one hour. The product was isolated as described in Example 2 to give the biologically active amino-6-demethyl-6-deoxytetracycline (B).

X 0.1N HCl 21g 263 350 ; 0.1N NaOH 241 27Q 385 max r max r

Example 8.

Preparation of nitro-6-deoxytetracycline.

The procedure of Example 1 was used except that 932 mg (2 m mol) of 6-deoxytetracycline hydrochloride [J. A. C. S. 80, 5324 (1958)], prepared by contacting a polar solvent solution of tetracycline with hydrogen in the presence of boric acid and a noble metal catalyst until about 1 mole of hydrogen had been absorbed for each mole of tetracycline, was dissolved with stirring in 50 ml of concentrated sulfuric acid at 0° C. 200 mg (2 mmol) potassium nitrate was gradually added to the dark brown reaction solution while stirring. After this addition, the solution became lighter as it turned dark yellow. Stirring of the reaction solution at 0°C was continued for about five minutes and then it was gradually poured into 1 liter of ether at 0°C with a stirring rate that kept the temperature between 0° and 2°C. A pale yellow solid that precipitated was filtered , washed with ether (4 x 100 ml) and dried under vacuum for two hours at room temperature. Weight 850 mg.

500 mg of the above nitration product was dissolved in 10 ml of water. To the dark brown solution was added sufficient of a concentrated solution of sodium carbonate to give the solution a pH of 5.0. A yellow birefringent solid that precipitated was filtered, washed with water (3 x 2 ml.) and dried under vacuum at 60° C. for two hours. Weight 260 mg. Melting point, chars at 198° C.

Analysis:

Calculated for C22H23N30!i.2H20 (509.4):

51.9% C, 5.34% H, 8.25% N,

7.06 percent H 2 O.

Found: 52.18% C, 5.15% H, 8.29% N,

4.41 percent H2O (loss during drying).

The ultraviolet spectrum of this

compound compared with 6-deoxytetracycline in 0.1N HCl shows a shift in the short wavelength from 269 m^i to 262 m^, and in the long wavelength from 345 m|i. to 360 m|j,. The infrared spectrum has a nitro absorption band at 6.55 µm and 7.43 µm

Example 9.

Preparation of amino-6-deoxytetracycline.

A solution of 10 mg of nitro-6-deoxy-tetracycline acid sulfate prepared as in Example 8 in 5 ml of water was stirred and an excess of zinc-copper was added. The pH was kept between 3 and 2 by the addition of 1.0N hydrochloric acid. The mixture was stirred for 15 minutes and the solution was filtered from excess zinc to give amino-6-deoxytetracycline

l 0.1N HC1 21? 263 346 x 0.1N NaOH 244 277 37Q max r max ' r

Example 10.

Production of nitro-6-deoxytetracycline sulfate.

A solution of 6-deoxytetracycline was prepared by slowly adding 1.00 g (0.0021 mol) of 6-deoxytetracycline hydrochloride to 40 ml of cold concentrated sulfuric acid. This solution was stirred and 216 mg (0.0021 mol) of potassium nitrate was added over a 5 minute period, the reaction flask being kept in an ice bath. The solution was stirred for 10 minutes and added more 216 mg of potassium nitrate as indicated above. After stirring for a further 25 min, the solution was slowly poured onto 400 g of ice while stirring. The resulting aqueous solution was extracted with n-butanol. The organic extract was washed with water and evaporated under vacuum until a precipitate formed. This sulfate salt was collected (0.75 g). Rr = 0.56 (n-butanol: pH 2 phosphate buffer).

Example 11.

Preparation of amino-6-deoxytetracycline disulphate.

A suspension of 645 mg of nitro-6-deoxytetracycline sulfate salt in 65 ml of ethanol with 65 mg of platinum dioxide and 3 drops of concentrated sulfuric acid was hydrated at room temperature and atmospheric pressure. The hydrogen uptake was 3 mol per moles of nitro-6-deoxytetracycline. The solution was filtered from the catalyst and evaporated to a small volume. Excess ether was added and the precipitated amino-6-deoxytetracycline disulfate was collected (600 mg). Rf = 0.03

(n-butanol: pH 2 phosphate buffer).

Example 12.

Preparation of amino-nitro-6-deoxytetracycline disulphate.

To a cold solution of 100 mg of amino-6-deoxytetracycline disulfate in 4.0 ml of concentrated sulfuric acid, 16 mg of potassium nitrate was added with stirring. The reaction flask was kept in an ice bath and the solution stirred for 5 minutes. After this time, it was added slowly with stirring to 100 ml of cold anhydrous ether. The solid was collected and washed with ether and then dissolved in anhydrous methanol. The solution was filtered, evaporated to a small volume and excess ether was added to precipitate the amino-nitro-6-deoxytetracycline disulfate salt. This was collected and washed with ether (65 mg). R{ = 0.45 (n-butanol: pH 2 f osph at-puf f er).

Example 13.

Preparation of bromo-nitro-6-deoxytetracycline sulfate.

A solution of 60.5 mg (0.1 m mol) bromo-6-deoxytetracycline sulfate (prepared by bromination of 6-deoxytetracycline in concentrated sulfuric acid with one equivalent of N-bromosuccinic acid) and 10.1 mg (0.1 m mol) potassium nitrate in 2.0 ml of concentrated sulfuric acid was stirred at ice bath temperature for 10 minutes. The solution was slowly poured into 50.0 ml of cold ether, separating 45 mg of solid. Rf = 0.81 (n-butanol; pH 2 phosphate buffer).

Example 14.

Production of bromo-nitro-6-deoxy-6-demethyltetracycline sulfate.

A solution of 59.1 mg (0.1 mmol) bromo-6-deoxy-6-demethyltetracycline sulfate (prepared by bromination of 6-deoxy-6-demethyltetracycline in concentrated sulfuric acid with one equivalent of N-bromosuccinimide) and 10.1 mg (0.1 mmol) of potassium nitrate in 2.0 mL of concentrated sulfuric acid was kept at ice bath temperature for 10 minutes. The reaction mixture was slowly added to 100 ml of cold ether. The solid that separated weighed 54 mg.

Example 15.

Preparation of amino-bromo-6-deoxy-6-demethyltetracycline disulphate.

An excess of zinc-copper preparation was added to a solution of 20 mg of bromo-nitro-6-deoxy-6-demethyltetracycline sulfate in 3.0 ml of water. The mixture was stirred at room temperature for 15 minutes during which time the pH of the solution was maintained at 3 by adding 0.1 N sulfuric acid as needed. The solution was filtered and the filtrate freeze-dried. The residue was triturated with methanol and filtered. The filtrate was evaporated to dryness under reduced pressure to yield 6.0 mg of material. R 1 = 0.21 (n-butanol; pH 2 phosphate buffer).

Example 16.

Preparation of 7,9-dinitro-6-demethyl-6-deoxytetracycline.

Powdered potassium nitrate (0.9 g - 0.009 mol) was added with stirring to 5.0 g (0.009 mol) 7-nitro-6-demethyl-6-deoxytetracycline hydrochloride in 100 ml. concentrated sulfuric acid. The solution was stirred for 45 minutes and then added dropwise with stirring to 2 liters of cold anhydrous ether. The solid was filtered and added to 1 liter of anhydrous ether, stirred for V2 hour, filtered and dried. The product was stirred in 1.7 liters of water and after the insoluble material had been removed by filtration, the filtrate was neutralized to pH 4.5 with 10% sodium hydroxide. The second precipitate was filtered, washed well with water and dried.

Patent Claim:

Process for the preparation of biologically active stable tetracycline compounds comprising substituted 6-deoxytetracyclines of the formula:

and acid addition salts thereof, where Ri or R2 means nitro and the other is hydrogen, nitro, amino or halogen, Ra is hydrogen or methyl and R4 is hydrogen or hydroxy, characterized in that a compound of the general formula I where Ri and/or R 2 is hydrogen, or an acid addition salt thereof, is reacted with a nitrating agent and then carried out if desired

a reduction of the nitro group to an amino group, and if Ri and R 2 in the starting material were both hydrogen, the amino compound formed is possibly reacted with a nitrating agent.

Claims (3)

Process for converting a gas mixture contaminated with sulfur compounds consisting of synthesis gas containing carbon monoxide and steam to hydrogen and carbon dioxide, wherein the gas mixture is brought into contact with a first catalyst at high temperature and wherein at least part of the resulting partially converted gas mixture is brought into contact with another catalyst at a relatively low temperature, characterized in that a first catalyst is used which comprises a chromium oxide-activated ferric oxide, said high temperature being in the range 316-538°C, the effluent from this step is cooled, after which at least part of the cooled effluent without any intermediate carbon dioxide removal is contacted with the second catalyst comprising a) at least one alkali metal compound derived from an acid having an ionization constant of less than 1 x 10^ and b) a hydrogenation-dehydrogenation component comprising at least one element selected from groups VB, VIB, and VIII in the periodic table, i the weight ratio between the hydrogenation-dehydrogenation component, calculated on the basis of its oxide, and the alkali metal compound, calculated on the basis of its oxide, is in the range from approx. 0.001:1 to approx. 10:1. 2. Method according to claim 1, characterized in that the gas mixture is brought into contact with the first catalyst in two stages. 3. Method according to claim 1 or 2, characterized in that the second catalyst is sulphided before use.