BE696488A - - Google Patents

Description

  

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  There NEW TETRACYCLINE DERIVATIVES AND THEIR PREPARATION "
The present invention relates to improvements and modifications to the process for the preparation of novel aminotetracyclines according to the main patent.



   There has been described in the main patent a process for the preparation of tetracycline derivatives containing an amino group in position 7 and / or in position 9 in which at least one of said amino groups is substituted by one or two radicals of formula:
 EMI1.1
 

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 wherein R3 and R4 are the same or different and represent hydrogen atoms, lower alkyl, aryl, substituted aryl, arylalkyl or substituted arylalkyl, said process comprising:

   a) reacting a tetracycline having an amino group in position 7 and / or in position 9 in which the amino group can be formed in situ, with one or two moles per amino group of a reagent for the introduction of a alkyl, arylalkyl or substituted arylalkyl, capable of forming a carbon-nitrogen bond on the nitrogen atom of the amine; b) and, if necessary, separately or simultaneously with step a), reacting the product of step a) with hydrogen to reduce an unsaturated carbon-nitrogen bond or to replace any radical with hydrogen. other than hydrogen on the alkyl portion of the substituted alkyl, arylalkyl or arylalkyl group;

   with the proviso that when there is an oxy-carbonyl atom on the carbon bound to the amino nitrogen, the product of step a) reacted with hydrogen, is only one
 EMI2.1
 7- and / or 9-formylaminotëtraGyolinej and, c) when two moles of alkylating or arylalkylating reagent are used per amino group, the second mole can be introduced separately or- simultaneously with the formation of the carbon / nitrogen bond and / or reaction with hydrogen.



   It has been found according to the present invention that when the process of the main patent is carried out using a 7-aminotetracycline, the product can

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 be a mixture of 7-monoalkylamino- and 7-dialkylamino-tetracyclines, and when using 9-amino-tetracycline, the product may be a mixture of 9-mono-alkylamino- and 9-dialkylaminotetracyclines.

   This is illustrated by Examples A to G below. Further, if a starting aminotetracyoline is used which has been prepared in such a way that a mixture of a 7-aminotetraoyeline and a 9-aminotetracyoline has been obtained, the process of the main patent has been carried out. *, with this mixture as raw material, can result in a corresponding mixture of products.



   The present invention relates to an improved process for the preparation of N-substituted aminotetracyalines in which the amino group is at position 7 of the tetracycline ring; the invention further relates to an improved means for obtaining a preferred compound belonging to the class of said 7- (aminosulbstituted) -tetracyclines, namely 7-dimethylamino-6-demethyl-6-deoxytetracycline, in a novel form. crystallized, stable.



   The embodiment of the present invention for obtaining 7- (amino-substituted) -teracyolines relates to a novel reductive alkylation process, for the preparation of compounds, of the following general formula;

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 EMI4.1
 wherein R6 is a hydrogen atom or a methyl group and R7 a mono (lower alkyl) -amino or di (lower alkyl-amino group. According to the present invention, the compounds corresponding to said general formula are prepared. by reacting a compound of general formula:

   
 EMI4.2
 in which R6 is a hydrogen atom or a methyl group, X a bromine, chlorine or fluorine atom, and R an aryl group - with a carbonyl compound of the general formula
 EMI4.3
 in which R1 is a hydrogen atom or a lower alkyl group and R2 is a hydrogen atom or a lower alkyl group, in the presence of a reducing agent, arylazo groups

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   (R-N-N- in the preceding formula) suitable for the implementation of the new process according to the present invention, can
 EMI5.1
 be for example groups:

   phenylazo, 2-sultophenylazo, 27 aiitrophenylazo, p-chlorophenylazo, -bomophenylazo, 2,5-di-chlorophenylazo, 2-naphthylazo, 1-naphthylazo, 2,4-dinitrophenylazo, 2-acetamidophenylazo, etc.



   It is understood that the term "lower alkyl" used in the present description covers all alkyl groups having up to about 6 carbon atoms. Consequently, the aldehydes and ketones useful in the implementation of the new reductive alkylation process according to the invention comprise, for example, formaldehyde, acetaldehyde, propionaldehyde, n-butyraldehyde, iso-butyraldehyde acetone, methyl ethyl ketone, diethyl ketone, etc.

   Typical raw materials to be used in the new process according to the invention include:
 EMI5.2
 7-phenylazo-Ila-bromo-6-deoxytetracycline, 7-p-cyanophenylazo-ila-bromo-6-deoxytetracycline, 7 - ehlorophenylazo-11a-bromo-6-deoxytetraayeline, 7;, phenylazo-21a-6-ehloro deoxytetraeyeline, 7-2-sulfophenylazo-Ila-chloro-6-deoxytetracycline,? -p-nitrophenylazo -ila-ehoro-6-deoxytetraoyeline, 7- ± -bromophenYlazo-11a-fluoro-6-deoxytetracyol- 1ne 7- ( dichlorophenylazo) -1, la-fluoro-6-dôsoxytetracyolin <µ 7- (2-naphthylazo) -ila-fluoro-6-deoxytetracyaline, .Ù / µf '7- (3-n1tro-4-methylphenylazo) -11a-bromo -6-demethyl-6- deoxytetracycline,

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 EMI6.1
 7- 2-nitro-4-ohlorophnylazo -.1, -b oto = 's. d:

   and ..-; .. deoxytetracycline, 7- (2-nitrophenylazc) -lla-bromo-6-demethj-l-f-deoxy- ¯ tetracycline,
 EMI6.2
 7-phenylazo-1la..ch3oro-6-demétaahyl = -dsoxytdtxscycii.ns 7-E-sulfophenylazo-.la-chloro-6-demety3.-6-deoxy-tetracycline, 7-E-nitrophdnylazo-12a..ehloro- 6-demethy.-6.ddsoxy- tetracycline, 7-n-carboxyphenylazo-lia-fluoro-6-demethyl-6-deoxy- tetracycline, 7-n-aoetamidophényiazo-ila-fiUoro-6-demethyi-6-deoxy- tetracycline , 7- (2,4-dinitrophenylazo) -lla-fluoro-6-demethyl-6-de-soxytetracyclines ,; and the like.



  The starting 7-arylazo-ila-halo-6-deoxy-tetracylines for the new process of the present invention can be prepared by processing a slurry.
 EMI6.3
 aqueous solution of an appropriate Ila-halo-6-deoxytetracycline by an aqueous solution or suspension of an aryldiazcum salt at a temperature of about -10 to about 50 C and in a pH range of about 4 to about 10. The exact conditions which are best in any given case will depend largely on the stability and reactivity of the diazonium salt used, as well as the pH of the reaction mixture.

   The aryl-diazonium salts suitable for use in the novel reductive alkylation process according to the present invention,

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 are essentially those which contain an electron attracting substituent such as:; nitro-cyanato, sulfo, cyano, carboxyl, thiocyanato or halo bonded to the aromatic ring ortho or para with respect to the diazonium function. By way of example of the corresponding arylamines, mention may be made of: aniline; p-chloroniline; p-bromaniline, 2,5-dichloraniline; 2,5-dibromaniline; p-nitroaniline; o-nitro-aniline, 3-nitro-4-toluidine; 2-nitro-4-chloraniline; 2,4-dinitroaniline; p-naphthylamine, α-naphthylamine; sulfanilic acid, benzidine and ao-to-p-phenylenediamine.



  Details regarding the preparation of the diazonium salts can be found in reference books such as "A.C.S. Monograph Series No. 127, The Chemistry of Synthetic Dyes", Rheinhold Publishing Corp., New York (1955) pp, 96-109.



     The essential characteristic of the new reductive alkylation process according to the invention lies in the specificity of the coupling reaction; the 7-phenylazo compound being formed exclusively, rather than a mixture of the 7- and 9-phenylazo compounds.

   Other means of synthesis, such as the nitration of 6-deoxytetracycline, as described in J.A.C.S. 82, 1253 (1960), by JH Bouthe et al., Lead to a mixture of 7- and 9-nitro isomers, The relative proportion of 7- and 9-nitro isomers produced by nitration is variable and depends to a certain extent, poorly understood, of the nature of 6-deoxytetracycline used as substrate; the absence or presence of a methyl group

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 in position 6 for example, strongly influencing the proportion of the two isomers produced.

   It is surprising to see that a similar mixture of isomers is not formed when a diazonium salt is coupled with a 6-deoxy-tetracycline,
Intermediate 11α-halo-6-deoxytetracyclines can be easily prepared by halogenation in position. 11a of the corresponding 6-deoxytetracylines, by the general methods described in the United States patent. No. 2,984,686.

   Suitable halogenating agents for use in this process include bromine, chlorine, N-chloro- and N-bromoamides of lower alkanoic acids, for example N-chloro- and N-bromo-. acetamides, imides of dicarboxylic acid, for example N-chloro and N-bromosuccinimide; N-lower alkanoyl-anilines, for example N-chloroacetanilide; 3-ohloro-, 3-bromo-, 3,5-dichloro and 3,5-dibromo-5,5-dimethylhydantoins; and lower alkyl hypochlorites. For the introduction of fluorine, perchloryl fluoride is used.



   The novel reductive alkylation process according to the invention can be carried out by chemical or catalytic reduction using processes which are well known to those skilled in the art. The catalytic reduction which is particularly suitable for the reductive alkylation of the aforementioned starting tetracycline can be carried out in a solvent for the starting tetracycline in the presence of a carbonyl compound and a metal catalyst and 'hydrogen

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 gaseous at pressures equal to or greater than atmospheric pressure. Usually, the reductive alkylation is conveniently carried out at hydrogen pressures of about 1 to 4 atmospheres.

   Temperature does not appear to be critical in the catalytic hydrogenation. Temperatures of 0 to 50 C and usually room temperature are preferred because they give the best results. The metallic catalyst may be a compound of a current metal such as nickel or drunk chromite, or a compound of a finely divided noble metal such as platinum, palladium or rhodium. The noble metal catalysts are advantageously used on a support such as finely divided alumina, activated carbon, diatomaceous earth etc .; and are commonly available in this form. The hydrogenation is carried out until the desired amount of hydrogen gas is absorbed and the hydrogenation is stopped at this point.

   The solvents chosen for the catalytic reaction must be inert to the reaction, i.e. they are not capable of reacting with the starting materials, the reaction product or the hydrochloride. drogenous under the reaction conditions. A variety of solvents can be used for this purpose and laboratory tests allow selection of an appropriate solvent for a particular starting tetracycline.

   In general, catalytic reductive alkylation can be carried out in solvents such as water, lower alcohols, for example methanol and ethanol; (lower alkoxy) -lower alkanols, for example

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 2-methoxyethanol, 2-ethoxyethanol, tetrahydrofuran, dioxane, dimethylformamide, etc.,
Various chemical reductants can be used in the reductive alkylation process as shown in the main patent,
Likewise, the products are obtained from the reductive alkylation reaction mixtures by standard methods, such as those described in the main patent.



   The characteristic compounds which can be prepared by the novel reducing alkylation process according to the present invention include, for example:
 EMI10.1
 7-methylamino-6-deoxy-6-demethyltetracycline, 7-diethylamino-6-deoxy-6-demethyltetracycline, 7-isopropylamino-6-deoxy-6-demethyltetracycline, 7-dimethylamin-6-deoxy-6-demethyltetracycline, 7-isopropylamino-6-deoxy-6-demethyltetracycline, 7-dimethylamino-6-deoxy-6-demethyltetracycline methyiamino-6-deoxytetracyoline, 7-diethylamino-6-deoxytetracycline, 7-Isobutylamïno-6-deoxytetracybline, and 7-di-n-butylam1no-6-deoxytetracycline.



   The tetracyclines according to the present invention are biblogically active and possess the broad spectrum antibacterial activity of the previous tetracyclines.
 EMI10.2
 known. In particular, 7-dimethylamino-6-deoxy-6-demethyltetracycline possesses extraordinary oral and parenteral activity against Staphylococcus aureus, strain of Smith,

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 and Stphylooocous aureus, Rose strain, responsible in particular for certain infections in mice. This activity is dealt with in detail in the main patent.



   The tetracyclines according to the invention are amphoteric compounds and therefore their acid addition salts can easily be prepared, i.e.
 EMI11.1
 mono- and di-functional salts. Preferred acids are the pharmaceutically acceptable, non-toxic acids, for example mineral acids such as hydrochloric acid and sulfuric acid, and the like, although organic acids such as trichloroacid can also be used. tick. Acid addition salts can be prepared by treating the novel tetracyclines with about 1 to 2 equivalents of the selected acid in an appropriate solvent.



   However, according to another embodiment, the present invention provides a crystalline shape.
 EMI11.2
 stable tallization of 7-dimethylamino-6t-demethyl-6-deoxyteracybline and more particularly, 7-dimethylamino-6-demethyl-6-deoxytetracyoline hydrochloride dihydrate and a new process for its preparation.



   This method is particularly useful for converting amorphous forms of 7-dimethylamino-6-demethyl-6-deoxytetracycline into a crystalline form. The method is also useful for improving the
 EMI11.3
 purity of 7-dimethylamino-6-demethyl-6-deoxytetracycline.

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  As a result, the product looks better, is more suitable for therapeutic application, and furthermore, the product is more stable. The more purified product is more suitable for therapeutic applications because the foreign material which may be present in some cases causes undesirable physiological side effects.



   7-Dimethylamino-6-demethyl-6-de-soxytetracycline is amphoteric, as it has both acidic and basic groups in its molecule and as a result, it sometimes reacts as an acid and sometimes as a base. In addition, it has some very special properties, due to a tendency to partial polymerization or rearrangements, in particular 4-epimerization which inactivates 7-dimethyl-
 EMI12.1
 amino-6-demeth, 1-6 - deoxytetraoyallne unless it is subjected to a particularly gentle treatment.



   Although, as indicated above
 EMI12.2
 Above, 7-dlmethylamino-6-demethl-6-deoxytetracycline could be produced in useful therapeutic forms, by the ordinary methods of crystallization from organic solvents, it was found that the yields are relatively poor, so that these processes, if used commercially, significantly increase the cost price of
 EMI12.3
 Therapeutically acceptable 7-dimeth, ylamino-6-d6-methyl-6-deoxytetracycline. In addition, significant 4-epimerization occurs when employing these methods. Adsorption chromatography gives a satisfactory product, but from a commercial point of view, adsorption chromatography is very

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 more inconvenient and expensive than the new method of the invention.



  It is an object of the present invention to prepare 7-dimethylamino-6-demethyl-6-deoxytracyclin in a therapeutically active form at the highest practical yield and as inexpensively and efficiently as possible. In addition, the present invention also relates to the preparation of 7-dimethyl- hydrochloride.
 EMI13.1
 arino- & demethyl-6 = deoxytetracyeline dihydrate with the least expensive and inconvenient modifications to the preliminary purification process.

   According to the process of the present invention it is possible to carry out the preliminary steps under identical conditions and to carry out the transformation into the desired final form shortly before the final isolation of the purified substance, so that 'a major modification of the process is not necessary to obtain the hydrochloride of 7-dimethylamino-6-demethyl-6-deoxytetracycline dehydrate,
According to the present invention, it is
 EMI13.2
 dissolves the amorphous and / or impure 7-dimethylamino-6-demdthyl-6-deoxytetracycline in water at a concentration between about 5% by weight and the saturation concentration.



  For best results, it is preferred to dissolve 7-di-
 EMI13.3
 methylamino-6-demethyl-6-deoxytetracycline in water at a concentration of about 20% / about 25% by weight. At this point the insoluble substances are separated by filtration and the solution can then be clarified, if desired, by treatment with activated charcoal, diatomaceous earth, etc.

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  The pH of the clarified solution is then adjusted to about 2 to 6 with hydrochloric acid or hydrochloric gas, after which the oristallization of 7-dimethylamino-6-demethyl-6-deoxytetracycline dihadrate hydrochloride begins.
The entire process is carried out at a solution temperature of about Ce to about 80 ° C .; but best results are obtained at a temperature of 20 to about 30 ° C. For best results, it is preferred to adjust the pH of the clarified solution to about 3.5-4.0.

   After the
 EMI14.1
 crystallization of 7-dimethyl-amino-6-demethyl-6-deoxytetracyeline hydrochloride dihydrate, the best results are obtained by maturing the suspension of the salt in solution at a temperature of about 0 to about 10 C for a period of time. - time period of about 1 hour. The product, the hydrochloride
 EMI14.2
 of 7-dimethylamino-6-demethyl-6-deoxytetraeycline dihydrate, is then recovered from the suspension by any suitable means such as filtration or centrifugation, and dried.



   It is quite surprising that 7-dimethylamino-6-demethyl-6-deoxytetraoyoline hydrochloride dihydrate so readily crystallizes in aqueous solution to give a product of high purity and activity, while other salts 7-dimethylamino-6-demethyl-6-deoxytetracycline such as sulfate, phosphate, maleate, tartrate and the like are not satisfactory due to either recovery in low yield, or a loss of activity due to 4-epimerization.

   In addition, even 7-dimethylamino-6-demethyl-6-deoxytetracycline dihydrochloride is absolutely unsatisfactory.

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 since it is an amorphous product. It is of particular interest that all other methods of crystallization of
 EMI15.1
 7-Dimethylamino-6-demethyl-6-deoxytetracyclil hydrochloride from organic solvents, for example, give products which are amorphous, impure or which, for other reasons, are not satisfactory for use in the forms of. administration.

   The new process of the present invention, further
 EMI15.2
 on the other hand, gives a 7-dimethylamino-6-demo-6-deoxytetraoyoline hydrochloride dihydrate of high purity, very easily and with little or no 4-epimerization,
7-Dimethylamino hydrochloride.



    6-Demethyl-6-deoxytetracycline dihydrate can be dehydrated in stages, first to monohydrate and then to anhydrous salt. However, both of these forms are hygroscopic and turn back to the dihydrate in a humid atmosphere.



   Examples A to G illustrate the preparation of (amino-substituted) -tetracyolines according to the process described in the main process patent in which mixtures of products are obtained.



  EXAMPLE A
 EMI15.3
 7wethlamlno and -8imeth-lamino-6-deoxv-6-demeth¯yl.ttetracyline A solution of 792.9 mg (1.5 milli-moles) of 7-amino * -6-deoxy * -6-methyltotracycline sulfatera in 90 cm3 of methyl cellosolve, 1.5 cm3 of 40% aqueous formaldehyde solution and 300 mg of a 10% palladium-on-charcoal catalyst, is hydrogenated at room temperature

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 and at atmospheric pressure. The uptake of 1 to 1.5 equivalents of hydrogen is complete after 1 to 2 hours. The catalyst is filtered off and the orange solution is poured into 1.5 liters of dry ether.

   The precipitate is separated by filtration, washed well with dry ether, and dried; it weighs 391 mg. Turbidimetric titration gives 852 gammas per cm3.



  83 mg of the material is chromatographed on neutral diatomaceous earth using a heptane-ethyl acetate-methanol-water partition system in the ratio of 45: 55 15: 6.



  The compound is in the form of the free base, the pH of the sample is 5. The product is eluted in the second retaining volume and weighs 15 mg;. It consists of a mixture of 7-methylamino and
 EMI16.1
 7-dimethyiamino-6-deoxi-6-demethyltetraoµelin.



  Analysis: Calculated for C22507N3: N: 9.45; N-CH 3 (3) 10,: 1.5.



   Found N: 9.71; N-CH3 (3): 10.04.



   Turbidimetric titration gives 219 gammas per cm3, R 0.65 (nitromethane: benzene: pyridines pH 3.4 buffer-20: 10: 3: 3).



  EXAMPLE B
 EMI16.2
 7-môthmlamino and -dimeth lamino - & - deox -6-demeth 1-tetrae - cline
A solution of 527 mg of sulfate
 EMI16.3
 of 7-amino-6-deoxy-6-demethyltetraoyoline, 0.25 cm3 of 97% formic acid and 0.2 cm3 of 37% formaldehyde solution, is heated at reflux for 2 hours. The mixture is cooled and taken up in 100 cm3 of water. Chromatography on

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 paper reveals the presence of 7-methylamino-and 7-dimethyla-
 EMI17.1
 m1no..6-deoxy-6- <1mthYltétac # ol1né'êt of their 4-epitlers.



  EXAMPLE CL 7-meth lamino-6-deox -6-demeth ltetrac eline A solution of 278.7 mg of sulphate of 7-Ritro-6 - = 'deoxy-6-demethyltetracyoline in 15 cm3 of memthyl cellosolve at 03, ce (7% water), 0.75 cm3 of 40% aqueous formaldehyde solution and 50 mg of 10% pallaidium-on-charcoal catalyst, is hydrogenated at ambient temperature and at atmospheric pressure. Fixation is complete (4 equivalents) after 2 hours. After removal of the catalyst
 EMI17.2
 by filtration, the solution is poured into 300 em3 of rec ether. The precipitate is collected by filtration, washed well with dry ether and dried; it weighs 200 mg. The product is a m-
 EMI17.3
 mixture of 7-methylamino- and 7-dimethylamino-6-esoxy-6-to ± methyltetracycline.

   Turbidimetric titration gives 100 gammas per cm3.



    EXAMPLE D
 EMI17.4
 9-methvlamino and -dimeth lamino-6-deox -6-demeth ltetrac eline A solution of 233.5 mg of 9-a hydrochloride; ino-6-deoxy-6-demethyl-tetracyeline in 8 cm3 of acid 0.1 N methanolic hydrochloric acid, 15 cm3 of methylcellosolve, 0.5 cm3 of 40% aqueous formaldehyde solution and 100 mg of 10% palladium-on-carbon catalyst, is hydrogenated at room temperature and at atmospheric pressure . The binding of 1 to 1.5 equivalents of hydrogen is complete in less than one hour. The catalyst is separated by

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 filtration and the solution is poured into 250 om of dry ether.



  The precipitate is washed well with dry ether and dried. It weighs 171 mg. The product is a mixture of 9-methylamino and 9-
 EMI18.1
 dimethylamino-6-d6soxy-6-demethyltetraoyoline, EXAMPLE E 9-meth lamino and -.dimeth lamino-6-dCSOx -6demeth I, Etracvc- A solution of 114.8 mg of 9-nitro-6-deoxy-6-ddmdthyltetracycline in 20 em3 of methanol containing 2.5 em3 of methanolic hydrochloric acid 0.1 N.fj 0 om 'of 40% aqueous solution of formaldehyde and 50 mg of 10% palladium on carbon catalyst, is hydrogenated at tem-. ambient temperature and atmospheric pressure. Fixation is complete (85% of theory) in one hour. After filtering off the catalyst, the solution is evaporated to dryness. The product obtained weighs 1.5 mg.

   The product is a mixture of
 EMI18.2
 9-methylamino and 9-dimethylamino-6-ddsoxy-6-demethyltetraoyellnes EXAMPLE y Z-ethvlamino and 1-diethylamino-6-deoxy-6-demethyltetracycline
A solution of 396 mg of 7-amino-6-deoxy-6-demethyltetracycline sulfate in 50 cm3 of methyl-cellosolve is hydrogenated at room temperature and at atmospheric pressure, using 2 cm3 of acetaldehyde and 150 mg of 10% palladium-on-carbon catalyst.



  The binding stops after 3 hours, the catalyst is separated by filtration, and the solution is poured into 750 cm3 of dry ether. The precipitate is separated by filtration, although

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 washed with dry ether and dried; it weighs 64 mg. The product is a
 EMI19.1
 mixture of 7-ethylamino and -diethylamino..6..deoxy-6-demQ-thyltetracycline, EXAMPLE G
 EMI19.2
 7-ethl - amina and -dieth lamino-6-deox -6-demeth ltetrae aline In 30 cm3 of methylcellosolve, 560 mg of 7jhnitro-6-deoxy-6-de-methyltetracycline sulfate, 2.1 cm3 of 1N sulfuric acid, 1 cm3 of acetaldehyde and 100 mg of 10% palladium on charcoal catalyst. The mixture is shaken with hydrogen for 1.5 hours, and the catalyst is then filtered off.



  The filtrate is poured into approximately 400 cm3 of ether and a slightly colored solid precipitates. After filtration and drying, the product weighs 182 mg. The product is a mixture of 7-ethylamino-
 EMI19.3
 and 7-d! .ethylamino-6-deoxy-6-demethyltetracycline,
The following examples illustrate the present addition, without however limiting its scope.



  Set 1
 EMI19.4
 L1a-chloro-6-demethyl-6-deoxxtetraeycline pret1on
A solution of 4.0 g (9.68 millimoles) of 6-demethyl-6-deoxyteracycline in 280 ml of methanol and 300 ml of benzene is evaporated to dryness at room temperature and under vacuum. A further 100 ml of ber., Zene is added to the residue and the mixture is again evaporated to dryness. The residual solid is immediately dissolved in the same flask in 120 ml of purified dioxane. This dioxane is purified in the manner

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 next; dioxane stored in calcium hydride is distilled over fresh calcium hydride.

   The fraction having a boiling range of 101 to 102 ° C. is collected. This subs. tance is free from peroxide and substantially anhydrous. To the solution of 6-demethyl-6-deoxytetracycline in dioxane, 1.416 g (10.6 millimoles) of N-chlorosuccinimide in 20 ml of dioxane are added. The mixture is stirred for 1 minute during which the potassium iodide test for the presence of chlorine becomes negative. The solution is freed from the solid formed by filtration. The filtrate is diluted with 1400 ml of Skellysolve C and stirred. The product is filtered, washed with Skellysolve C and dried at 35 C and 2.5 mm Hg for about 10 hours to give 3.9 g of the.
 EMI20.1
 l1a-chloro-6-demethyl-6-deoxytetracycline with a yield of 90%.



  Example 2
 EMI20.2
 Preparation of 11a-chloro-6-deoxytetraccline
A total of 1.617g (3.8 millimoles) of 6-deoxytetracycline, dried as described in Example 1, is dissolved in 50 ml of purified dioxane. A solution of 0.555 g (4.18 millimoles) of N-chlorosuccinimide dissolved in 8 ml of dioxane is added. The mixture obtained is treated exactly as described in Example 1 to give., 5g
 EMI20.3
 ila-chloro-6-deoxytet-racycline, with a yield of 86.5%.

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    Example
 EMI21.1
 luoration of 6-demeth 1-6-deox tetraocline in position 1Âa
A solution of 4.15 g (0.01 mol) of 6-demethyl-6-deoxytetraoycline in 250 ml of methanol and 40 ml of water is obtained when the slurry is treated with 15 ml of a 0.787N solution. of sodium methoxide in methanol (0.0118 mol). The solution is cooled to 0 ° C. in an ice-salt bath, which causes the formation of a light fine precipitate. Perchloryl fluoride was vigorously bubbled through the stirred liquid under a nitrogen atmosphere for 20 minutes, during which time the pH fell to 7.

   An aliquot of the reaction mixture shows a ratio of
 EMI21.2
 absorptions at 271 mu and 354 mau of 615 (in O, IN acid), indicating that the fluorination is substantially complete. The excess perchloryl fluoride is removed with nitrogen and the reaction mixture is evaporated in vacuo with gentle heating to about 50 ml. The pH of the relatively clear solution was adjusted to 4.5, and an abundant precipitate formed. After maturing in a shaker for 2 hours, the solids are filtered off and washed successively with 10 ml of cold water, with cold methanol and 6 times with ether. After drying under vacuum, 2.53 g of light brown substance are obtained.



    Example 4
 EMI21.3
 Copulation of 11a-chloro-6-demethxl-6-deoxytetracycline with the diazonium salt of sulfanilic acid A total of 3.0 g (6.7 millimoles) of 11a-chloro-6siemethyl-fi is suspended. -deoxytetra-

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   cyclin in 150 ml of water. The suspension is brought to pH 7.5 with approximately 3.5 ml of 0.5N NaOH. The suspension is stirred at 0-5 C for about 30 minutes, The diazonium salt of sulfanilic acid is prepared as follows, a solution of 0.39 g (3.68 millimoles) of anhydrous sodium carbonate in 27.5 ml of water. 1.408 g (7.35 millimoles) of sulfanilic acid monohydrate is added and the mixture is heated to effect dissolution.

   The solution is cooled to 0-5 C and then mixed with 0.555g (8.0 millimoles) of sodium nitrite dissolved in 3 ml of water.This mixture is poured into 7.7 g of crushed ice containing 1, 5 ml (18.8 mil- limoles) of concentrated HCl and the mixture was kept in an ice bath. The white crystals of the precise diazonium salt pite from this mixture. The pH of the suspension of lia-chloro-6-
 EMI22.1
 6-demethyl-deoxyt6tracycline is readjusted to 7.5 with 0.5N NaOH, then 1 ml of 0.5N Na 2 CO 3 solution is added to raise the pH to 8.5. Slowly add the suspension of the diamond salt while the pH is maintained at 7.5 by addition of Na2CO3, 0.5N. Total solution is obtained and stirred for about 2 hours at 0-5 C.

   The ice bath is then removed and the solution is acidified to 2.7 with concentrated HCl.
 EMI22.2
 



  The brown solid obtained, 11acloro-6-dmethyl-6-dsoxy-7-p-sUlfophénylazotétracyol1ne, is centrifuged, washed with water and dried at 3500 under 2.5 mm for 16 hours to give 2.831 g. free acid, with a yield of 66%.

 <Desc / Clms Page number 23>

 



  Example 5
 EMI23.1
 Copulation of Ila-chloro-6-deoxytetracycline with the diazonium salt of sulfanilic acid
A total of 1.5 g (3.34 millimoles) of 11a-chloro-6-deoxytetracyoline is suspended in 72 ml with / water. The suspension is adjusted to pH 7.5 / approximately 3.8 ml of NaOH,
0.5N. Most of the substance goes into solution after stirring at 0-5 C for about 20 minutes. We prepare a
 EMI23.2
 solution of 0.189g (1.75 millimoles) of Na2CO3 in 13.4 ml of water, 0.68g (3.54 millimoles) of sulfanilic acid monohydrate is added to this solution and the mixture is heated to effect the dissolution. This solution was cooled to 0-5 C and then mixed with 0.268 g of NaNO 2 (3.98 millimoles) dissolved in 2 ml of water.

   This mixture is poured into 3.8 g of crushed ice containing 0.75 ml (8.2 millimoles) of concentrated HCl and kept in an ice bath. White crystals of the diazonium salt precipitate from this mixture. We readjust the pH
 EMI23.3
 of a solution of lla-ohloro-6deoxytetraoycline at 7.7 with NaOH, 0.5 !, 5.5 ml of 0.5N Na2CO3 solution are then added to bring the pH to 8.5. The diazonium salt suspension is added slowly and the pH is maintained at 7.5 with 5N Na2CO3. This mixture is stirred for 1 1/2 h at 0-5 C. The ice bath is then removed and the solution is acidified to pH 1.5 with concentrated HCl. The solid is centrifuged, transferred into a funnel, washed with water and dried at 35 ° C. under 2.5 mm for
 EMI23.4
 16 hours.

   The dried product, 11a-chloro-6-deoxy-7-p-sulfophenylazotetracycline weighs 0.937g.

 <Desc / Clms Page number 24>

 



  Example 6
 EMI24.1
 Copulation of li, a..ohloro-6-deoxytetracycline with the diazonium salt of -nltrniline ¯ ¯¯¯, ¯¯¯¯
A suspension of 0.484 g of p-nitraniline in 22 ml of water and 0.76 ml of concentrated HCl is prepared.



  1/2 g of crushed ice is added and the mixture is maintained at 0-5 C using an ice bath. Then added to the suspension of p-nitraniline 0.27 g of NaNO 2 dissolved in 5 ml of water. The! most of the solid goes into solution. This solution of the diazonium compound of p'nitraniline is added.
 EMI24.2
 Slowly added to a solution of 1.5 g of 11a-chloro-6-deoxy-tetracycline. The populating solution is stirred at 0-5 C for about 30 minutes. The ice bath is removed and the pH is brought to 3.5 with concentrated HCl. The solution is diluted with an equal volume of water.

   The product that formed, lla-chloro-
 EMI24.3
 6-deoXY-7-p-nitrophenylazotetracycline, is centrifuged, transferred to a funnel, washed with water and dried at 350 ° C. under 2.5 ml for 16 hours to give 1.68 g of product, yield 84. 5%.



  Example 7
 EMI24.4
 Copulation of 11a-fluorine-6-demethyl-6-deoxytetracyoline with the diazonium salt of sulfanilic acid
A slurry of diazotized sulfanilic acid is prepared by dissolving 0.48 g (0.0025 mole) of sulfanilic acid and 0.133 g of anhydrous sodium carbonate in 5 ml of water by the action of heat. The solution is cooled, 0.19 g of sodium nitrite is added and then the mixture is poured.

 <Desc / Clms Page number 25>

 solution in 2.5g of ice and 0.5g of concentrated HCl contained in a beaker placed in an ice bath.

   The diazonium salt precipitates and is added dropwise as a slurry) at 0-5 C to a cold solution of 0.66g (0.002 mol)
 EMI25.1
 of iia-riuoro-6-demethyl-6-dexoxytetracycline in 15 ml of 0.5N sodium carbonate solution and 4 ml of 0.5N NaOH.



  The vessel is closed and stirred in an ice bath for 2 hours at 0-2 ° C. The pH is adjusted to 2.7 and the mixture is stirred for 15 minutes at room temperature. The solid substances which have precipitated after adjusting the pH are separated by filtration and washed with a small amount of water and then a 4: 1 mixture of ether-methanol. After drying, the product weighs 1.08 g.



  Example 8
 EMI25.2
 Reductive alkylation of lla-chloro-6-demethyl-6-deoxy-7-p-sulfophenylazotetracycline to 6-dem4thyl-6-deoxy -? - dimô-thylaminotetracycline
We suspend 2g of lla-chlo-
 EMI25.3
 ro-v-demethyl-6-deoxY-7-p-sulfophenylazotetraoycline in 40 ml of methanol and 6 ml of 2N H2SO4. Ig of palladium is added to
 EMI25.4
 5% on carbon (humidity at 50) and 7 ml of formaldehyde (37%).



  The mixture was reduced in a Parr shaker for 2 hours at 26 ° C. The total amount of hydrogen absorbed was 6.9 kg. The catalyst is filtered off and washed with 5 ml of methanol. We. slowly add the filtrate to a beaker containing 800 ml of acetone previously cooled to -10 C. The precipitated light yellow solid is filtered off, washed with 2 portions

 <Desc / Clms Page number 26>

 of 5 ml of acetone and dried at 35 ° C. under 2.5 mm for about 16 hours to give 1.55 g of the product with a yield of 79.5%.



  Example 9
 EMI26.1
 Reductive alkylation of Ila-chloro-6-dimethyl-6-d6soxy-7-p-sulfophenylazotetraoyoline to 6-demethyl-6-deoxY-7-dîmêthyl-aminot6tracMeline ¯¯¯¯¯¯¯¯¯¯¯¯¯¯ <# <#, 19 of 11a-chloro-6-demethyl-6-deoxy-7-p-sulfophériy.azotetracyeline is suspended in 1 ml of methanol and 3 ml of H2SO4, 2N, 0.5 g of 5% oatalyst is added of palladium on carbon (50% wet). The mixture is reduced in a Parr seoousses apparatus at 27.5 C. The reduction is complete in 1 hour during which 2.26 kg of hydrogen have been resorbed. The catalyst is then filtered off and washed with 1 ml of methanol. 0.5 g of fresh catalyst and 3.5 ml of formaldehyde (37%) are added to the filtrate.

   Reduction is continued for a further hour during which the mixture absorbs a further 2.44 kg of hydrogen. The catalyst is filtered with 1 ml of methanol. The filtrate and the washing liquor are slowly added to 400 ml of acetone previously cooled to -10 C. The very light yellow precipitate is filtered off, washed with 3 ml of cold acetone and dried at 35 ° C. under 2.5. mm for 16 hours, to give 0.76 g of product with a yield of 76%.



  Example 10
 EMI26.2
 Reductive alkylation of lla-chloro-6-deoxY-7-p-nitrophenyl-azotetrae cline to 6-deox - -dimeth laminotetrac cline 1 g of 1.1a-chloro-6-deoxY-7-p-nitroph6nylazotetracycline is suspended in 25 ml of methanol

 <Desc / Clms Page number 27>

 and 3 ml of H2SO4, 2N, 0.5 g of catalyst containing 5% palladium on carbon (50% wet) is then added and the procedure of example 9 is followed. The solution of 7-dimethylamino-6-deoxytetracycline is poured into 500 ml of cold acetone (at -10 C), The product is very soluble in acetone.

   Ether is then added and the precipitate is filtered off, washed with ether and dried at 35 ° C. under 2.5 mm for 16 hours to give 0.512 g of product (yield 48%).



  Example 11 Reductive methylation of 7-amino-6-deoxy-6-demethylated
 EMI27.1
 tracycline to give 7-dimethylamino-6-deoxy-6-demethyl-tutracycline
A solution of 792.9 mg (1.5 milli-
 EMI27.2
 mole) of? -amino-6-desoxy-6-demethylt; etraeycline sulfate in 90 ml of methylcellosolve, 1.5 ml of 40% aqueous formaldehyde solution 1 and 300 mg of 10% palladium catalyst on charcoal, is hydrogenated at room temperature under atmospheric pressure. The absorption of about 1.5 equivalents of hydrogen is complete in 1 to 2 hours. The catalyst is separated by filtration and the orange solution poured into 1.5 l of anhydrous ether. The precipitate is separated by filtration, washed well with anhydrous ether and dried; it weighs 391 mg.

   Turbidimebraic analysis gives 852 gammas per ml. 83 mg of the substance was chromatographed on neutral diatomaceous earth using a heptane-ethyl acetate-methanol-water system in the ratio 45: 55: 15: 6. We obtain the compound

 <Desc / Clms Page number 28>

 as the free base, the pH of the sample is 5.0.



  The product is eluted in the second holding volume, and weighs 15 mg.



   Turbidimetric analysis gives
 EMI28.1
 2190 gammas per ml, Rr 0.65 (nitromethane: benzene: pyridine: buffer pH 3.4 - in the proportion of 20: 10: 3: 3).



    Example 12 Reductive methylation of 7-amino-6-deoxy-6-demethyl-t-
 EMI28.2
 tracycline to give 7-dimethylamino-6-deoxy - & - demethyl-, tetracycline.



   A solution of 527 mg of 7-amino-6-deoxy-6-demethyltetraoycline sulfate, 0.25 ml of 97% formic acid and 0.2 ml of formaldehyde solution is refluxed for 2 hours. at 37%. The cooled mixture is taken up in 100 ml of water. Chromatography on paper reveals the pre-
 EMI28.3
 Sente of 7-dimethylamino-6-dexoxy-6-demethyltetracycime and its 4-epimer.



  Example 13 Reductive methylation of 7-nitro-6-deoxy-6-demethyltetra-
 EMI28.4
 cyclin to give 7-dimethylamino-6-deoxy-6-demethyititracycline.



   A solution of 278.7 mg of sulfate
 EMI28.5
 of 7-nitro-6-desoxy-6-demethyltetraeyeline in 15 ml of 93% methylcellosolve (7% water), 0.75 ml of 40% aqueous formaldehyde solution and 50 mg of 10% catalyst of palladium on charcoal, is hydrogenated at room temperature under atmospheric pressure. Absorption is total (4 equivalents) in 2 hours.

   After separation of the catalyst by filtration,

 <Desc / Clms Page number 29>

 the solution is poured into 300 ml of anhydrous ether, The precipitate is collected by filtration, washed well with anhydrous ether and seo; it weighs 200 mg, Example 14 Preferred method for the preparation of 7-dimethyl hydrochloride.
 EMI29.1
 thmino-6-demeth1-6-deoxetracyc11ne dihydrate
7 g (15 milli-moles) of 7-dimethylamino-6-demethyl-6-deoxytetracycline base are suspended in 15 ml of water (pH 5.85) and 15 ml (15 mmoles) of 1N HCl are added. . At pH 4.6 the solution is substantially clear, and at pH 4.4 precipitation begins. The final pH is 3.3.

   A dark-colored semi-solid substance floating on the surface of the reaction mixture is separated and it is found that the latter has a high content (6.2%) of epimer. The product is matured for 1 hour at 0 C, then collected by filtration and dried under 0.003 ml at 25 C.

   Yield 5.62 g (79%). This product and the starting material give the following analysis:
 EMI29.2
 
<tb> Substance <SEP> of
<tb>
<tb> departure <SEP> Product
<tb>
<tb> biological <SEP> analysis <SEP> -------------- <SEP> 955 <SEP> # / mg .------ <SEP> 940 <SEP> # / mg.
<tb>
 
 EMI29.3
 epimer ----------------- 17 '/ mg .------- 8 * / mg.
 EMI29.4
 
<tb> isomer <SEP> ----- <SEP> 9- <SEP> ------- <SEP> trace <SEP> ---------- <SEP> 0
<tb>
   Example 15 Preparation of crystallized 7-dimethylamino-6-demethyl-6-deoxytetracycline hydrochloride dihydrate.



   500 mg (1.1
 EMI29.5
 millimole) of 7-dimethylamind-6-demethyl-6-deoxytetracycllne

 <Desc / Clms Page number 30>

 base in 1 to 2 ml of 0.1N HCl and add 3N HCl until the pH reaches 2.6. The crystallized precipitate which formed is matured for 30 minutes at room temperature and then for 15 minutes at 0 C, collected by filtration and dried. Yield 280 mg (52%).



    Example 16
Process for the purification of 7-dimethylamino-6- hydrochloride
 EMI30.1
 demé $ rl-6-déscx tetrae nline dihydrate
About 900 mg of the residual product of the preparation of crystallized 7-dimethylamino-6-demethyl-6-deoxytetracycline dihydrate monohydrochloride is suspended in 3 ml of water and the pH is adjusted to 1.3 with HCl 1, 0N. "Darco" is added to the resulting solution and after 15 minutes the mixture is filtered through Celite no. 512. The filtrate is adjusted with NaOH to pH 3.7 and a crystalline precipitate then separates out. The mixture is cooled in an ice bath and the precipitate is collected, washed with acetone and dried. Yield 540 mg (60%).



  Example 17 Hydration of 7-dimethylamino-6-demethyl-6- hydrochloride
 EMI30.2
 de? xytétra.cyo 1 ine monohydrate
The speed and the degree of water absorption at 24 C are measured on a sample of hydrochloride
 EMI30.3
 of 7-dimethyiamino-6-demethyl-6-deoxytetracycline containing 3.8% water (monohydrate). For a thin film sample, the equilibrium moisture content is reached within 20 to 30 minutes.

 <Desc / Clms Page number 31>

 
 EMI31.1
 
<tb> Humidity <SEP> relative <SEP> Content <SEP> in <SEP> H2O <SEP> at <SEP> equilibrium
<tb> 15% <SEP> 7.48 <SEP>%
<tb>
<tb> 19% <SEP> 7.56 <SEP>% <SEP>
<tb>
<tb> 44% <SEP> 8.25 <SEP>% <SEP>
<tb>
 
The water contents at equilibrium are slightly higher than the theoretical value (6.98%) for a dihydrate. Moisture absorption is reversible.

   The equilibrium sample has a relative humidity of 44%, rapidly losing weight at 20% relative humidity to a final moisture content of 7.64%.



  Example 18 Preparation of crystallized 7-dimethylamino-6-demethyl-6-deoxytetracycline hydrochloride dihydrate
15 g of 7-dimethylamino-6-demethyl-6-deoxytetracycline hydrochloride is added to 45 ml of water and the dissolution is completed by addition of concentrated HCl. The solution is treated with Darco and Magnesol and filtered.



  The pH of the filtrate is raised to 4.0 with sodium hydroxide. The crystallized precipitate is collected, washed with water, and dried at 25 ° C. and about 8% relative humidity. This product and the starting material give the following analysis:
 EMI31.2
 
<tb> Substance <SEP> of
<tb>
<tb> start¯¯¯ <SEP> Product
<tb>
<tb>
<tb>
<tb>
<tb> epimer <SEP> ---------- <SEP> 21.5 <SEP> # / mg <SEP> ------- <SEP> 11.0 <SEP> / mg .
<tb>
<tb>
<tb>
<tb>
<tb> isomer <SEP> 9- <SEP> ------- <SEP> 0 <SEP> --------------- <SEP> 0
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> content <SEP> in <SEP> H2O <SEP> --------------------------------- <SEP > 6.85%
<tb>
 

 <Desc / Clms Page number 32>

   Example 19
 EMI32.1
 Preparation of bis sulfate (7-dimethylamJno-6-demethyl-6- deox tetrae eline ¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯, In 2 ml of 'water,

   500 mg of 7-dimethylamino-6-demethyl-6-deoxytetracycline disulfate are dissolved. The solution is treated with a few mg of magnesol and filtered.



  The pH of the filtrate is raised to 3.9 with 5N NaOH. The crystallized precipitate which formed is collected and dried (yield 10.6 mg).



  Raising the pH to 6.5 causes precipitation of an amorphous product; thus the pH is adjusted to 4.0 and the solution is concentrated to about half of its volume. By ripening at 4 ° C., this solution gives approximately 6 mg of crystalline product.



  Raising the pH to 6.5 causes the separation of 36 mg of amorphous product, the yield of bis- (7-dimethylamino-6-demethyl-6-deoxytetracycline) sulfate is too low for this process to have a any practical application.



  Example 20 Preparation of 7-dimethylamino6-demethyl-6-esoxy-tetracycline maleate ¯¯¯¯¯
A mixture of 0.443 g is dissolved
 EMI32.2
 (0.1. Millimole) of -dimethylamino-6-demethyl-6-deoxytetra-cyclin base and 0.116 g (0.1 millimole) of maleic acid in water and the solution is brought to siocity in a rotary evaporator. 40 C. A 0.40 g portion of the solid residue is dissolved in 12 ml of absolute ethanol under reflux and the solution is filtered and maintained at -10 ° C. overnight. The solid

 <Desc / Clms Page number 33>

 which formed was collected by filtration, washed with ethyl ether and dried at 25 ° C. under 0.002 ml. The product (0.21 g) = 53%), appears to be crystallized under the polarizing microscope.



    - The product and the starting substance give the following analysis:
 EMI33.1
 
<tb> Substance <SEP> of
<tb> departure <SEP> Product
<tb> biological <SEP> analysis <SEP> --------- <SEP> 1020 <SEP> # / mg <SEP> -------- <SEP> 978 <SEP> # / mg
<tb>
<tb> epimer <SEP> ------------ <SEP> 12.6 <SEP> # / mg <SEP> --------- <SEP> 160 <SEP> # / mg
<tb>
 Example 21 Preparation of 7-dimethylaminq-6-demethyl-6-deoxytetracycline hydrochloride
In 15 ml of chloroform, dissolve
 EMI33.2
 500 mg of '-dimethylamino6-demethyl-6-deoxytetracycline and hydrogen chloride gas is bubbled through the solution. A yellow precipitate forms which is collected by filtration, washed with chloroform and dried. The product is amorphous.



  Example 22 Preparation of 7-dimethylamino-6-demethyl- hydrochloride
 EMI33.3
 6-deoxytetracycline ¯
In 10 ml of acetone, dissolve
500 mg of 7-dimethylamino-6-demethyl-6deoxy-tetracycline and a stoichiometric amount of concentrated hydrochloric acid is added to the solution. It separates an amorphous product. The addition of water and then acetone results in the separation of a crystalline product.

 

Claims (1)

CLAIMS EMI34.1 - ..a-a - -a -x..s ..xs 1. A process for the preparation of 7- (amino-substim>;) tetracycline of formula; EMI34.2 in which R6 represents a hydrogen atom or a methyl group and R7 a mono (lower alkyl) ami @@ or di (-lower alkyl) amino group, process characterized in that a compound of formula : EMI34.3 (in which X represents a bromine, chlorine or fluorine atom, R6 a hydrogen atom or a methyl group, and R an aryl group) with a carbonyl compound of the formula: EMI34.4 <Desc / Clms Page number 35> (where R and R 2 are the same or different and each represents a hydrogen atom or a lower alkyl group) in the presence of a reducing agent. 2 - A process according to claim 1, characterized in that a compcsé is used in which X is a chlorine atom and R1 and R2 are hydrogen atoms. 3 A method according to claim '. or 2, characterized: in that a compound is used in which R is a p-nitrophenyl or p-sulfophenyl residue. 4 - A method according to one of the preceding claims, characterized in that a compound is used in which R6 is a hydrogen atom. 5 - A process according to one of claims 3 to 3, characterized in that a compound in which R6 is a methyl group is used, 6 - A process according to one of the preceding claims. teeth characterized in that a compound is formed in which R7 is a dimethylamino group. 7 - A method according to one of the preceding claims characterized in that one uses as agent re- EMI35.1 i, (i produces hydrogen with a hydrogenation catalyst based on a noble metal. 8 - A method according to claim 7, characterized in that the catalyst consists of palladium or platinum distributed on an inert support, <Desc / Clms Page number 36> 9 - A method according to claim 8, characterized in that the catalyst is a palladium on carbon catalyst. 10 - A process for the purification of 7-dimethylamine EMI36.1 no-6-demethyl-6-aesoxytetracycline produced for example by the process according to one of the preceding claims 1 to 4 or 6 to 9, characterized in that sufficient 7-di- EMI36.2 impure methylamino-6- <ieinethyr-de-oxytetraoyoline in water to achieve a concentration between about 5% by weight and the saturation concentration; any Insoluble substance is separated; the pH of the clarified solution is adjusted EMI36.3 at a temperature of about 0 to 80 ° C at a pH of about 2 to about 6 by aialo? kzydr3n¯ued acid following which 7-dimcithylamlno6dérnethyl.6deo; ytétxaoycline crystallizes as hydrochloride dehydrate2 and then the 7-dir!) ethylamino-u "demethyl-6-deoxytetraoyoline dihydrate hydrate is recovered. II - A method according to claim 10, characterized in that the concentration of the solution is from about 20% to about 25% by weight, 12 - A method according to claim 10 or 11, characterized in that the pH is adjusted to 3.5-4.0. 13 - A method according to one of revendioations 10 to 12 characterized in that the temperature is between about 20 and 30 C. <Desc / Clms Page number 37> EMI37.1 14 - 7-Dimethylamino-6-demôthyl-6> * de3oxytetracyoline hydrochloride monhydrate. 15 - 7-Dimethylamino-6-deneethyl-deso-zrtetraoylene hydrochloride dihydrate.